Ca2+- and Protein Kinase C-dependent Signaling Pathway for Nuclear Factor-κB Activation, Inducible Nitric-oxide Synthase Expression, and Tumor Necrosis Factor-α Production in Lipopolysaccharide-stimulated Rat Peritoneal Macrophages

Abstract

Lipopolysaccharide (LPS)-activated macrophages are pivotal in innate immunity. With LPS treatment, extracellular signals are transduced into macrophages via Toll-like receptor 4 and induce inflammatory mediator production by activating signaling pathways, including the nuclear factor-κB (NF-κB) pathway and the mitogen-activated protein kinase (MAPK) pathway. However, the mechanisms by which the intracellular free Ca2+ concentration ([Ca2+]i) increases and protein kinase C (PKC) is activated remain unclear. Therefore, we investigated the signaling pathway for Ca2+- and PKC-dependent NF-κB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-α (TNF-α) production in LPS-stimulated rat peritoneal macrophages. The results demonstrated that the LPS-induced transient [Ca2+]i increase is due to Ca2+ release and influx. Extracellular and intracellular Ca2+ chelators inhibited phosphorylation of PKCα and PKCβ. A PKCβ-specific and a general PKC inhibitor blunted phosphorylation of serine in mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) 1. Moreover, a MEKK inhibitor reduced activation of inhibitoryκB kinase and NF-κB. Upstream of the [Ca2+]i increase, a protein-tyrosine kinase inhibitor reduced phosphorylation of phospholipase C (PLC) γ. Furthermore, a PLC inhibitor eliminated the transient [Ca2+]i increase and decreased the amount of activated PKC. Therefore, these results revealed the following roles of Ca2+ and PKC in the signaling pathway for NF-κB activation in LPS-stimulated macrophages. After LPS treatment, protein-tyrosine kinase mediates PLCγ1/2 phosphorylation, which is followed by a [Ca2+]i increase. Several PKCs are activated, and PKCβ regulates phosphorylation of serine in MEKK1. Moreover, MEKKs regulate inhibitory κB kinase activation. Sequentially, NF-κB is activated, and inducible nitric-oxide synthase and tumor necrosis factor-α production is promoted. Lipopolysaccharide (LPS)-activated macrophages are pivotal in innate immunity. With LPS treatment, extracellular signals are transduced into macrophages via Toll-like receptor 4 and induce inflammatory mediator production by activating signaling pathways, including the nuclear factor-κB (NF-κB) pathway and the mitogen-activated protein kinase (MAPK) pathway. However, the mechanisms by which the intracellular free Ca2+ concentration ([Ca2+]i) increases and protein kinase C (PKC) is activated remain unclear. Therefore, we investigated the signaling pathway for Ca2+- and PKC-dependent NF-κB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-α (TNF-α) production in LPS-stimulated rat peritoneal macrophages. The results demonstrated that the LPS-induced transient [Ca2+]i increase is due to Ca2+ release and influx. Extracellular and intracellular Ca2+ chelators inhibited phosphorylation of PKCα and PKCβ. A PKCβ-specific and a general PKC inhibitor blunted phosphorylation of serine in mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) 1. Moreover, a MEKK inhibitor reduced activation of inhibitoryκB kinase and NF-κB. Upstream of the [Ca2+]i increase, a protein-tyrosine kinase inhibitor reduced phosphorylation of phospholipase C (PLC) γ. Furthermore, a PLC inhibitor eliminated the transient [Ca2+]i increase and decreased the amount of activated PKC. Therefore, these results revealed the following roles of Ca2+ and PKC in the signaling pathway for NF-κB activation in LPS-stimulated macrophages. After LPS treatment, protein-tyrosine kinase mediates PLCγ1/2 phosphorylation, which is followed by a [Ca2+]i increase. Several PKCs are activated, and PKCβ regulates phosphorylation of serine in MEKK1. Moreover, MEKKs regulate inhibitory κB kinase activation. Sequentially, NF-κB is activated, and inducible nitric-oxide synthase and tumor necrosis factor-α production is promoted. Lipopolysaccharide (LPS), 2The abbreviations used are: LPS, lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor α; TLR4, Toll-like receptor 4; TRAF, TNF receptor-associated factor; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; MEKK, mitogen-activated/extracellular signal-regulated kinase kinase; MEK, mitogen-activated/extracellular signal-regulated kinase kinase kinase; IKK, IκB kinase; PKC, protein kinase C; PTK, protein-tyrosine kinase; IκB, inhibitory κB; TAK, transforming growth factor β-activated kinase; TAB, TAK1-binding protein; [Ca2+]i, intracellular free Ca2+ concentration; Ins(1,4,5)P3, inositol 1,4,5-triphosphate; iNOS, inducible nitric-oxide synthase; PLC, phosphatidylinositol-specific phospholipase C; Fura-2/AM, Fura-2 acetoxymethyl ester; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; PtdIns(4,5)P2, phosphatidylinositol 4,5-biphosphate; DAG, 1,2-diacylglycerol; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. 2The abbreviations used are: LPS, lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor α; TLR4, Toll-like receptor 4; TRAF, TNF receptor-associated factor; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; MEKK, mitogen-activated/extracellular signal-regulated kinase kinase; MEK, mitogen-activated/extracellular signal-regulated kinase kinase kinase; IKK, IκB kinase; PKC, protein kinase C; PTK, protein-tyrosine kinase; IκB, inhibitory κB; TAK, transforming growth factor β-activated kinase; TAB, TAK1-binding protein; [Ca2+]i, intracellular free Ca2+ concentration; Ins(1,4,5)P3, inositol 1,4,5-triphosphate; iNOS, inducible nitric-oxide synthase; PLC, phosphatidylinositol-specific phospholipase C; Fura-2/AM, Fura-2 acetoxymethyl ester; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; PtdIns(4,5)P2, phosphatidylinositol 4,5-biphosphate; DAG, 1,2-diacylglycerol; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. one of the membrane components of Gram-negative bacteria, induces a variety of responses to severe infection, such as local inflammation, antibody production, and septic shock. Macrophages respond to LPS early in the infection and thus play a pivotal role in host defense. However, at high concentrations, LPS stimulates macrophages to release massive amounts of pro-inflammatory mediators such as interleukins (ILs), tumor necrosis factor α (TNF-α), superoxide, and nitric oxide (1Su G.L. Am. J. Physiol. 2002; 283: G256-G265Crossref PubMed Scopus (422) Google Scholar, 2dos Santos C.C. Slutsky A.S. J. Appl. Physiol. 2000; 89: 1645-1655Crossref PubMed Scopus (413) Google Scholar). These pro-inflammatory mediators can disturb normal cellular function, and this disruption can lead to multiple organ dysfunction syndromes or lethal septic shock (3Marshall J.C. Crit. Care Med. 2001; 29: S99-S106Crossref PubMed Scopus (358) Google Scholar, 4Shen F.M. Guan Y.F. Xie H.H. Su D.F. Shock. 2004; 21: 556-560Crossref PubMed Scopus (47) Google Scholar). Therefore, the investigation of LPS-induced signaling pathways in macrophages is important and necessary for discovering potential therapeutic targets and drugs. In recent years, it has been found that LPS binds with LPS-binding protein and interacts with a complex that consists of CD14, MD2, and Toll-like receptor 4 (TLR4); the complex transduces the signal generated by its interaction with LPS into the interior of the cell (5Mancuso G. Midiri A. Biondo C. Beninati C. Gambuzza M. Macri D. Bellantoni A. Weintranb A. Espevik T. Teti G. Infect. Immun. 2005; 73: 5620-5627Crossref PubMed Scopus (61) Google Scholar, 6Matsuguchi T. Masuda A. Sugimoto K. Nagai Y. Yoshikai Y. EMBO J. 2003; 22: 4455-4464Crossref PubMed Scopus (77) Google Scholar). The intracellular Toll/IL-1 receptor domain of TLR4 recruits myeloid differentiation factor 88, IL-1 receptor-associated kinase, myeloid differentiation factor 88 adaptor-like protein, Toll/IL-1 receptor adaptor protein, and TNF receptor-associated factor 6 (TRAF6) to form a complex, which can transduce the signal further. Several signaling pathways are activated sequentially, including the mitogen-activated protein kinase (MAPK) pathway and the nuclear factor-κB (NF-κB) pathway (7Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3357) Google Scholar). In LPS-stimulated macrophages, MAPKs are activated by a three-tiered cascade of kinases: 1) mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK); 2) MAPK kinase (MEK); 3) MAPKs, including p38, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinase (JNK). MAPKs can activate some transcription factors, such as activator protein 1 and activating transcription factor 2, and play important roles in the production of inflammatory mediators (8Chi H.B. Barry S.P. Roth R.J. Wu J.J. Jones E.A. Bennett A.M. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2274-2279Crossref PubMed Scopus (462) Google Scholar, 9Liu Y.W. Chen C.C. Tseng H.P. Chang W.C. Cell Signal. 2006; 18: 1492-1500Crossref PubMed Scopus (92) Google Scholar, 10Gertsch J. Schoop R. Kuenzle U. Suter A. FEBS Lett. 2004; 577: 563-569Crossref PubMed Scopus (169) Google Scholar, 11Wang X.M. Kim H.P. Song R.P. Choi A.M.K. Am. J. Respir. Cell Mol. Biol. 2006; 34: 434-442Crossref PubMed Scopus (128) Google Scholar). However, how does LPS activate the MAPK pathway in macrophages? Some reports have suggested that phosphoinositide 3-kinase, protein kinase C (PKC), and some protein-tyrosine kinases (PTKs) are responsible for MEKK activation (12Greenberg S.S. Ouyang J. Zhao X.F. Wang J.F. Alcohol. 1998; 16: 167-175Crossref PubMed Scopus (26) Google Scholar, 13Hsu H.Y. Hua K.F. Lin C.C. Lin C.H. Hsu J. Wong C.H. J. Immunol. 2004; 173: 5989-5999Crossref PubMed Scopus (144) Google Scholar). NF-κB regulates the expression of many cytokines in LPS-stimulated macrophages and plays an important role in the immune system. Recently, considerable progress has been made in determining the function and regulation of NF-κB. In macrophages, NF-κB is a p65/p50 dimer and is inactivated by binding with inhibitory κB (IκB). The inhibitory κB kinase (IKK) β, a component of the IKK complex, can phosphorylate IκBα, and leads to the ubiquitination and degradation of IκB. Meanwhile, p65/p50 is translocated into the nucleolus (14Chen F. Castranova V. Shi X.L. Demers L.M. Clin. Chem. 1999; 45: 7-17Crossref PubMed Scopus (654) Google Scholar, 15Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar). Therefore, the activation of the IKK complex plays a crucial role in NF-κB activation. Furthermore, two sets of adaptors are linked to IKK activation. The first involves transforming growth factor β-activated kinase 1 (TAK1) and two associated adaptors, TAK1-binding protein 1 (TAB1) and TAB2. After stimulation, TRAF6 ubiquitylates itself or the component of the TAK1·TAB1·TAB2 complex and primes it for IKK activation. Another protein that has been reported to act as a bridge between TRAF6 and IKK is ECSIT. Moreover, MEKK1 and MEKK3 have been implicated in IKK activation in receptor/TLR4-mediated NF-κB activation pathways (7Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3357) Google Scholar). Although many studies have revealed that TRAF6 is related to MEKKs and IKK activation, the question about how TRAF6 activates them is unanswered. Intracellular free Ca2+ concentration ([Ca2+]i) is important for cellular function; however, few studies have been focused on the signaling pathway(s) involving [Ca2+]i in LPS-stimulated macrophages. Several reports have indicated that LPS treatment can cause an increase in [Ca2+]i, which is related to TNF-α production in alveolar macrophages and Kuffer cells (16Seabra V. Stachlewitz R.F. Thurman R.G. J. Leukocyte Biol. 1998; 64: 615-621Crossref PubMed Scopus (55) Google Scholar, 17Wheeler M. Stachlewitz R.F. Yamashina S. Ikejima K. Morrow A.L. Thurman R.G. FASEB J. 2000; 14: 476-484Crossref PubMed Scopus (110) Google Scholar). Our previous reports have also revealed that the transient increase in [Ca2+]i in LPS-stimulated rat peritoneal macrophages plays an important role in TNF-α production (18Wang H. Dong Z.Y. Yu Y. Liu B.H. Ma T. Xu J. Yang W.X. Acta Biophys. Sin. 2002; 18: 345-349Google Scholar). Furthermore, LPS also induces a biphasic change in inositol 1,4,5-triphosphate (Ins(1,4,5)P3) in C3H/HeN mouse macrophages, and the change leads to the activation of a TNF-α gene. The later phase of the Ins(1,4,5)P3 change is mediated by a γ2 type of phosphatidylinositol-specific phospholipase C (PLCγ2) (19Shinji H. Akagawa K.S. Tsuji M. Maeda M. Yamada R. Matsuura K. Yamamoto S. Yoshida T. J. Immunol. 1997; 158: 1370-1376PubMed Google Scholar). However, the mechanisms by which LPS induces a transient increase in [Ca2+]i and the role of [Ca2+]i in NF-κB and MAPK signaling pathways have not been elucidated. Additionally, LPS can activate PKC isoenzymes and regulate the expression of inflammatory mediators in macrophages (20Fronhofer V. Lennartz M.R. Loegering D.J. J. Leukocyte Biol. 2006; 79: 408-415Crossref PubMed Scopus (22) Google Scholar). However, which kinds of PKC isoenzymes are responsible for NF-κB activation and how they work in the signaling pathway remain unknown. In the present study, we investigated the [Ca2+]i- and PKC-dependent signaling pathway for NF-κB activation, inducible nitric-oxide synthase (iNOS) expression, and TNF-α production in LPS-stimulated rat peritoneal macrophages. On the basis of the experimental results, we propose a new mode of the signaling pathway. Reagents—Dimethyl sulfoxide, LPS (Escherichia coli serotype 0127:B8 prepared by phenol extraction), Fura-2 acetoxymethyl ester (Fura-2/AM), the PTK inhibitor (genistein), the PKC inhibitor (GF109203X), the PLC inhibitor (U73122), and EGTA were purchased from Sigma. BATPA-AM (intracellular Ca2+ chelator) was obtained from Molecular Probes. The MEKK inhibitor (PD98059), the PKCα-specific inhibitor (Go6976), and the PKCδ-specific inhibitor (Go6983) were purchased from Calbiochem. The PKCβ-specific inhibitor (LY379196) was acquired from Eli Lilly. Isolation and Culture of Macrophages—Male Wistar rats (about 250 g), which were treated humanely in compliance with institutional guidelines, were killed. Hanks’ balanced salt solution was injected into the abdomen of each rat. Macrophages in Hanks’ balanced salt solution removed from the abdomen were centrifuged at 200 × g for 5 min. The cell pellet was collected, and the cells were cultured in RPMI 1640 and 10% fetal bovine serum for 6 h at 37°C in 5% CO2. Adherent cells were harvested, re-suspended, and incubated for another 48 h before analysis. Nonspecific esterase staining showed that 95% of the adherent cells were macrophages. Immunoblot Assay to Detect PLC Phosphorylation—Cells (107) were scraped from dishes into cold lysis buffer (0.1 m Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 20 mm NaF, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride (PMSF), and 1 μg/ml each of pepstatin and leupeptin) after they were washed twice in cold phosphate-buffered saline (PBS). After centrifugation (10,000 × g for 10 min at 4 °C), the soluble fraction, which contained PLC, was collected. The proteins in the soluble fraction were separated by SDS-PAGE (7%) and transferred to polyvinylidene fluoride membranes. Immunoblotting was carried out by treating the membrane with 4.5 μg of antibody specific for activated phosphotyrosines or phosphoserines of PLCγ1 (Tyr783), PLCγ2 (Tyr759), or PLCβ3 (Ser537) (Cell Signaling Technology; ratio of antibody dilution, 1:200) for 12 h. To normalize the amount of sample in each lane, immunoblotting was also carried out by using 4.5 μg of polyclonal antibody to PLCγ1, PLCγ2, or PLCβ3 (Cell Signaling Technology; ratio of antibody dilution, 1:200) for 12 h. Biotinylated antibody specific for rabbit immunoglobulin (Amersham Biosciences) was employed as the secondary antibody. The chemiluminescence of the streptavidin-horseradish peroxidase (HRP) conjugates was detected by film. Measurement of [Ca2+]i in Macrophages—Intracellular free Ca2+ was detected by using the ratiometric fluorescent Ca2+ indicator dye Fura-2 and a microspectrofluorometer. Cells were incubated in 3 μm Fura-2/AM for 50 min at room temperature and washed twice with PBS. Changes in the fluorescence intensity of Fura-2 at excitation wavelengths of 340 and 380 nm and the emission wavelength of 510 nm were monitored in an individual peritoneal macrophage. [Ca2+]i was calculated by using the following equation.[Ca2+]i≈Kd×[Ca-Fura-2][Fura-2]≈Kd×F340F380(nmol/l) Kd was the constant for Fura-2 chelating Ca2+, and its value was about 135 nmol/liter when the temperature was 22 °C. In the present experiment, F340/F380 was directly related to [Ca2+]i. Measurement of Total PKC Activity—Total protein kinase C activity was determined as described by Lee and Wu (21Lee H.Z. Wu C.H. Eur. J. Pharmacol. 2000; 403: 195-202Crossref PubMed Scopus (4) Google Scholar) but with modifications. After treatment, cells were washed twice with PBS and scraped into cold lysis buffer (20 mm Tris-HCl, pH 8.0, 0.5 mm EDTA, 0.5 mm EGTA, 2.5 mm PMSF, 5 μg/ml leupeptin, and 5 μg/ml antipain). After collection, cells were sonicated for 10 pulses. Sonicated samples were centrifuged at 15,000 × g for 30 min at 4 °C, and the supernatant was collected and aliquoted (200 mg/tube). PKC activity in the supernatant was measured immediately by using the Pierce Colorimetric PKC Assay Kit. The PKC-dependent phosphorylated peptide was quantified by spectroscopy at 570 nm. Immunoblot Assay to Detect Phosphorylated PKC Isoenzymes—After treatment with LPS, macrophages were washed twice in ice-cold PBS. Cell pellets were resuspended in buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EGTA, 1 mm dithiothreitol, 1 mm PMSF, 1 mm Na3VO4, 1 mm NaF, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml antipain, 1% Nonidet P-40, 0.25% sodium deoxycholate) for 30 min at 4 °C. Lysates were centrifuged at 10,000 × g for 30 min at 4 °C, and the supernatants were collected. Proteins in the supernatant were separated by SDS-PAGE (12%). SDS-separated proteins were equilibrated in transfer buffer (50 mm Tris, pH 9.0–9.4, 40 mm glycine, 0.375% SDS, 20% methanol) and electrotransferred to Immobilon-P membranes. Nonspecific binding sites were blocked by incubation for 1 h in Tris-buffered saline (10 mm Tris, 150 mm NaCl) containing 5% nonfat dry milk and 0.05% Tween 20. The membranes were then washed and incubated with 6 μg of anti-phospho-PKCα (Ser657) antibody (dilution ratio, 1:5000)(Santa Cruz Biotechnology), anti-phospho-PKCβ (Ser660) antibody (dilution ratio, 1:2500) (Santa Cruz Biotechnology), anti-phospho-PKCδ (Thr505) antibody (dilution ratio, 1:1000) (Cell Signaling Technology), or anti-phospho-PKCε (Ser729) antibody (dilution ratio, 1:2500) (Upstate) for 10 h at 4 °C. To normalize the amount of sample in each lane, immunoblotting was carried out by treating the membrane with 6 μg of polyclonal antibodies to PKCα (dilution ratio, 1:5000), PKCβ (dilution ratio, 1:2500), PKCδ (1:500), and PKCε (dilution ratio, 1:500) (Santa Cruz Biotechnology) for 10 h at 4 °C. An HRP-conjugated goat anti-mouse IgG antibody (1:20,000 dilution) was used as the secondary antibody, and the chemiluminescence was detected on film. Immunoprecipitation and Immunoblot Analysis of Phosphorylated Serine in MEKK1—Cells were washed in ice-cold PBS and scraped into PBS. Cells were pelleted by centrifugation and lysed in whole cell extract lysis buffer (50 mm Tris-HCl, pH 7.9, 150 mm NaCl, 1.5 mm MgCl2, 1% Triton X-100, 10% glycerol, 1.0 mm EDTA, 1 mm Na3VO4, 1 mm PMSF, 0.2 mm leupeptin, 0.2 mm pepstatin A) for 30 min on ice. The lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and supernatants were isolated. After normalization (immunoblotting assay), cell lysates that contained equal amounts of total protein were incubated with 2 μg of polyclonal anti-MEKK1 antibody (43-Y, Santa Cruz Biotechnology; dilution ratio, 1:200) for 2.5 h at 4 °C and then with 4.5 μg of protein G-conjugated agarose beads (Amersham Biosciences) for 1 h at 4 °C. After centrifugation, MEKK1 isolated from the beads was separated by SDS-PAGE (12%) and transferred onto a nitrocellulose membrane. Immunoblot analysis was carried out by using monoclonal antibody to phosphoserine (Santa Cruz Biotechnology; dilution ratio, 1:1000). Biotinylated antibody to rabbit Ig (Amersham Biosciences) was employed as the secondary antibody. The chemiluminescence of the streptavidin-HRP conjugates was detected by film. Immunoprecipitation and IKK Protein Kinase Activity Assays—After LPS stimulation of macrophages, the cytosol was extracted by using 200 μl of IKK CE buffer (10 mm HEPES-KOH, pH 7.9, 250 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 0.2% Tween 20, 2 mm dithiothreitol, 1 mm PMSF, 20 mm β-glycerophosphate, 10 mm NaF, 0.1 mm Na3VO4), and the proteins in each extract were using from were incubated with 1 μg of monoclonal antibody specific for for 2 h at 4 °C, and then with protein G-conjugated agarose beads (Amersham Biosciences) for 1 h at 4 °C. After the beads were washed twice with IKK CE buffer and with kinase buffer (20 mm pH mm NaCl, 10 mm MgCl2, 2 mm dithiothreitol, 1 mm PMSF, 20 mm β-glycerophosphate, 10 mm NaF, 0.1 mm Na3VO4), they were incubated with 20 μl of kinase buffer containing 20 μm 10 of and 0.5 μg of at 30 °C for 30 min. The SDS-PAGE and the proteins were To normalize kinase the proteins that to were transferred to polyvinylidene fluoride membranes (Amersham and the immunoblotting to IKK D. A. 2005; PubMed Scopus Google Scholar). Immunoblot Assay to Detect Phosphorylated in the (107) were washed twice with 5 of cold PBS and centrifuged at × g for 5 min at 4 °C. Proteins were by of the pellet in 250 μl of buffer (10 mm Tris-HCl, pH 10 mm 2.5 mm 1.5 mm MgCl2, 1 mm Na3VO4, 0.5 mm dithiothreitol, mm 2 μg/ml leupeptin, 2 μg/ml pepstatin and 2 μg/ml The was incubated on for 10 min and then at a The centrifugation at × g for 5 min at 4 °C, and the supernatant was collected. After the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes and after the binding sites on the membranes were blocked by incubation at 4 °C with 1% bovine serum the membranes were then incubated with antibody specific for (dilution ratio, Cell for 12 h at 4 °C. To normalize the amount of sample in each lane, analysis was also carried out by treating the membranes with 5 μg of polyclonal antibody to (dilution ratio, Cell for 12 h at 4 °C. Biotinylated antibody to rabbit immunoglobulin (Amersham Biosciences) was employed as the secondary antibody. The chemiluminescence of the streptavidin-HRP conjugates was detected by film. of in (107) were washed twice with cold PBS. were by the pellet in 250 μl of buffer A (10 mm Tris-HCl, pH 10 mm 2.5 mm 1.5 mm MgCl2, 1 mm Na3VO4, 0.5 mm dithiothreitol, mm 2 μg/ml leupeptin, 2 μg/ml pepstatin and 2 μg/ml After incubation for 10 min on the was at a were collected by centrifugation at × g for 5 min at 4 °C and resuspended in μl of buffer A to 20 mm Tris-HCl, pH mm 20% were then lysed at 4 °C for 30 min with was by centrifugation at 10,000 × g for 30 min at 4 °C, and the supernatant was collected. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. After binding sites were blocked by incubation at 4 °C with 1% bovine serum the membranes were then incubated with polyclonal antibody specific for (Santa Cruz To normalize the protein of each was detected with μg of antibody 1:1000). Biotinylated antibody specific for rabbit immunoglobulin (Amersham Biosciences) was employed as the secondary antibody. The chemiluminescence of the streptavidin-HRP conjugates was detected by J.J. R. J. 2001; PubMed Scopus Google Scholar). and was by using a for Macrophages were with 3 μg of that contained an NF-κB and 3 μg of After 48 samples were and prepared for to the and were measured in each sample using the Assay Immunoblot to Detect LPS treatment, macrophages were lysed in sample buffer mm Tris-HCl, pH SDS, 10% glycerol, 5% and for 5 min at °C. (20 μg in a 10% and the proteins were transferred to nitrocellulose membranes were treated with 10% nonfat milk for 1 h to binding and incubated with rabbit polyclonal antibody to (Santa Cruz Biotechnology) at 4 °C. To normalize the protein of each was detected with μg of antibody 1:1000). The membranes were then treated with HRP-conjugated IgG (dilution ratio, for 1 h. were detected as described E.A. J. Infect. 1999; PubMed Scopus Google Scholar). TNF-α and cells and were cultured in TNF-α was detected in each After the an equal of with or was to the at of cell were collected and with 1 to centrifugation at 200 × g for 5 min. The supernatants were and 1 phenol was After 4 the supernatants were and μl of a solution of and was to each The were measured by an and the of TNF-α was calculated using a for TNF-α M. Y. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were a of and results from a are Some results are as the LPS-induced a in [Ca2+]i, PKC Serine in IKK, and NF-κB and TNF-α 5 min of the of LPS treatment (10 phosphorylated in PLCγ1 and PLCγ2 was detected in rat peritoneal macrophages, and phosphorylation a at 10 min. phosphorylation of PLCβ3 was not detected Moreover, a transient increase in [Ca2+]i was after the of LPS treatment and for a total of The of [Ca2+]i was when 5 mm EGTA was extracellular Ca2+ concentration was the of Ca2+ increase were in the and of EGTA Therefore, the results that the transient increase in [Ca2+]i is due to intracellular Ca2+ which followed by Ca2+ from the extracellular Furthermore, total PKC activity between and 1 h and then to The of change in the of phosphorylated isoenzymes β, and was to the in total PKC activity by LPS (10 in the phosphorylation of serine in which was first detected at h and for the h. 1 to 3 MEKK1 was phosphorylated Moreover, IKK activity the of 1 to 3 h and then decreased of phosphorylated in the and of into the nucleolus also in the first 3 h of LPS treatment Meanwhile, the of NF-κB activation in to LPS treatment of phosphorylated and expression