Stoichiometry of a Ligand-gated Ion Channel Determined by Fluorescence Energy Transfer

Abstract

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged α1-, β2-, or γ2-subunits of the hetero-oligomeric γ-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the α1(c-Myc)β2γ2 and the α1β2(c-Myc)γ2 receptors was twice that obtained with α1β2γ2(c-Myc), suggesting that there are 2× α-, 2× β-, and 1× γ-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining α1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2, but no significant energy transfer was observed with α1β2γ2(c-Myc) receptors, indicating the absence of a second γ-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, α1β2γ2, is composed of two copies each of the α- and β-subunits and one copy of the γ-subunit (i.e.(α1)2(β2)2(γ2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins. We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged α1-, β2-, or γ2-subunits of the hetero-oligomeric γ-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the α1(c-Myc)β2γ2 and the α1β2(c-Myc)γ2 receptors was twice that obtained with α1β2γ2(c-Myc), suggesting that there are 2× α-, 2× β-, and 1× γ-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining α1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2, but no significant energy transfer was observed with α1β2γ2(c-Myc) receptors, indicating the absence of a second γ-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, α1β2γ2, is composed of two copies each of the α- and β-subunits and one copy of the γ-subunit (i.e.(α1)2(β2)2(γ2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins. γ-aminobutyric acid, type A γ-aminobutyric acid monoclonal antibody europium cryptate minimum essential medium fetal calf serum Understanding the assembly and molecular interactions of proteins is a rapidly growing field in biology. Many techniques have been used to probe the interaction of proteins including, for example, immunoprecipitation, yeast two-hybrid analysis, circular dichroism, and more recently fluorescence energy transfer between two proteins in close proximity (1Mathis G. Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar, 2Thomas D.D. Carlsen W.F. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5746-5750Crossref PubMed Scopus (158) Google Scholar, 3Damjanovich S. Bene L. Abdelkrim A. Goldman C.K. Sharrow S. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13134-13139Crossref PubMed Scopus (120) Google Scholar). We have used the technique of fluorescence resonance energy transfer to investigate the stoichiometric assembly of a GABAA1receptor, a member of the major inhibitory ligand-gated ion channel family in the brain. It is proposed that the GABAA receptors are hetero-oligomeric pentamers composed of α, β, and a third subunit type, γ, δ, or ε. This is based on an analogy with other ligand-gated ion channels such as the nicotinic acetylcholine receptor (4Raftery M.A. Hunkapiller M.W. Strader C.D. Hood L.E. Science. 1980; 208: 1454-1457Crossref PubMed Scopus (360) Google Scholar, 5Cooper C.E. Couturier S. Ballivet M. Nature. 1991; 350: 235-238Crossref PubMed Scopus (402) Google Scholar), their size as measured by gel filtration and sucrose density centrifugation (6Mamalaki C. Barnard E.A. Stephenson F.A. J. Neurochem. 1989; 52: 124-134Crossref PubMed Scopus (31) Google Scholar, 7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar), and their image under electron microscopy (8Nayeem N. Green T.P. Martin I.L. Barnard E.A. J. Neurochem. 1994; 62: 815-818Crossref PubMed Scopus (378) Google Scholar). To date, 15 GABAA subunits have been cloned (α1–6, β1–4, γ1–3, δ, and ε), and expression studies reveal that for the most characterized subtypes at least one α, one β, and one γ-subunit are required to recover a fully functional GABAA receptor (9Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1150) Google Scholar). We have investigated the stoichiometry of the most abundant GABAAreceptor isoform in the brain, α1β2γ2, which is responsible for at least some of the therapeutic effects of benzodiazepines, barbiturates, and steroids (10McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1085) Google Scholar). Receptors modified with a c-Myc epitope on either the α1-, β2-, or γ2- subunit were expressed transiently in HEK293 cells. It has previously been shown that receptors transiently transfected in these cells using the same methods as employed here are appropriately assembled into αβγ-hetero-oligomers and are expressed on the cell surface. Other subunit assemblies do not reach the cell surface and are retained in the endoplasmic reticulum (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). mAbs to the c-Myc epitope were derivatized with the fluorescence donor, europium cryptate (EuK), or the fluorescent energy acceptor, XL-665. Europium, encaged by cryptate, emits a strong long lived fluorescent signal at 620 nm when illuminated with light at 337 nm from a nitrogen laser, which can be time-resolved from short lived background fluorescence. In addition to being used as a single label, analogous to a radiolabel for example, EuK also serves as an energy donor. The fluorescent signal produced by EuK can be transferred to an acceptor molecule if it is in close enough proximity. The recipient molecule for this fluorescent resonance energy transfer is a modified allophycocyanine, XL665, which fluoresces at 665 nm (for review of the homogeneous time-resolved fluorescence technology see Mathis (1Mathis G. Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar)). The transfer of energy from europium cryptate-labeled c-Myc antibodies to XL-665-labeled antibodies is therefore indicative of the two antibodies being in close proximity. Because the energy transfer is 50% at a distance of 9.5 nm (1Mathis G. Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar), this would require that the antibodies are very closely associated, for example in the same macromolecular complex. We used EuK-labeled c-Myc mAbs to quantify the number of subunits present on intact cells expressing α1β2γ2 subunits where each of the subunits was epitope-tagged with c-Myc and compared this with the number of receptors present using conventional radioligand binding. Fluorescence energy transfer was then used to confirm the stoichiometry of the receptor as (α1)2(β2)2(γ2)1. [methyl-3H]Ro15-1788 (87.0 Ci/mmol) was from NEN Life Science Products, Hertfordshire, United Kingdom. Flunitrazepam, GABA, and fetal calf serum were from Sigma. Minimal essential medium (MEM) was from Life Technologies, Inc. The GABAAreceptor α1-subunit was epitope-tagged by site-directed mutagenesis using methods described previously (13Wingrove P.B. Wafford K.A. Bain C. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4569-4573Crossref PubMed Scopus (216) Google Scholar). The epitope sequence (EQKLISEEDL) was introduced between Glu34and Leu35 (these two residues becoming the first and last amino acid of the epitope tag), just C-terminal to the putative signal peptide cleavage site. The β2 and γ2GABAA receptor subunits were epitope-tagged using a modified version of the pcDNA1Amp eukaryotic expression vector (pcDNA1AmpSignalMyc). This vector was constructed from the α1 c-Myc cDNA described above and contains the 5′-untranslated region of bovine GABAAα1-subunit (GenBank accession no. X05717), the signal peptide and six amino acids of the mature α1-subunit, and the c-Myc epitope tag sequence (EQKLISEEDL (14Evan G.I. Lewis G. Ramsay G. Bishop J.L. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar)) followed by a small polylinker into which the mature polypeptide of the subunit of interest can be inserted. The human β2-subunit c-Myc construct contains amino acids Glu38–Asn474 of β2, and the human γ2-subunit c-Myc construct contains amino acids Tyr46–le467. All constructs were confirmed by DNA sequencing. Constructs were prepared using polymerase chain reaction to generate the appropriate β2 and γ2 sequences, which were then subcloned into the pcDNA1AmpSignalMyc vector. DNAs were prepared for transfection by CsCl centrifugation. Transient transfection into HEK293 cells was performed exactly as described previously in detail using a 1:1:1 ratio of α1, β2, and γ2 cDNAs (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 15Wingrove P.B. Thompson S.A. Wafford K.A. Whiting P.J. Mol. Pharmacol. 1997; 52: 874-881Crossref PubMed Scopus (95) Google Scholar). The benzodiazepine site of the GABA receptor was labeled by the antagonist [3H]Ro15-1788, a radioligand frequently used to quantify receptors, because only fully assembled αβγ heterotrimers bind this ligand (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar, 12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16Duggan M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar). Nonspecific binding was determined using 10 μmflunitrazepam. Binding to the GABA binding site was carried out with [3H]muscimol, and 100 μm GABA was used to define nonspecific binding. Radioligand binding assays were carried out in a total volume of 0.5 ml in 10 mmKH2PO4, 100 mm KCl, pH 7.4, for 1 h at 4 °C prior to termination through Whatman GF/C filters followed by 3× 3-ml washes with cold assay buffer and scintillation counting. In binding studies carried out at one concentration of [3H]Ro15-1788, this was 1.8 nm. GABAA receptors solubilized from stably transfected cells were immunoprecipitated using anti-α1-, β-, or γ2-subunit antisera bound to protein A-Sepharose (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar). 50 μl of polyclonal antiserum were incubated with 50 μl of packed protein A beads in a total volume of 1 ml of Tris-buffered saline for 1 h at room temperature. Receptors were solubilized from the cells using a deoxycholate/Triton buffer (1% Triton X-100, 0.5% deoxycholate, 0.1 mm KCl, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 100 mm Tris-HCl, pH 8.2) by mixing cell membranes for 1 h at 4 °C with a detergent buffer at a protein concentration of 1 mg/ml. Aliquots (500 μl) of detergent-solubilized cell membranes were incubated with antibodies immobilized on protein A beads overnight at 4 °C. The receptor immobilized on protein A beads was washed three times with Tris-buffered saline/0.1% Tween 20 by centrifugation and resuspension and was finally resuspended in 0.5 ml of Tris-buffered saline, and 10–50-μl aliquots of packed beads were used for [3H]Ro15-1788 binding. The monoclonal anti-c-Myc mAb, 9-E10 (ATCC no. CRL-1729, Ref. 14Evan G.I. Lewis G. Ramsay G. Bishop J.L. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar) was used for these experiments. An antibody was purified from hybridoma supernatant by Cymbus Biotech, UK, and this was then derivatized with either the fluorophore, europium cryptate (at an average ratio of 9 molecules of europium cryptate per antibody molecule), or with the fluorescence acceptor, XL665 (at a ratio of 1 molecule of XL-665 per antibody molecule), by Cis-Bio International, Marcoule, France. Labeling of c-Myc mAb with EuK at this ratio gave 418 counts of fluorescence/s/fmol of antibody. Intact cells, transiently transfected with GABAA receptors with c-Myc-tagged subunits, were harvested by scraping, washed once by centrifugation (20 s at 1,000 rpm in a bench top Eppendorf Microcentrifuge), and resuspended in MEM + 5% FCS. [3H]Ro15-1788 binding (1.8 nm) was carried out to determine receptor density on intact cells. Cells containing the equivalent of 100 fmol of receptor were incubated with various concentrations of europium cryptate-derivatized mAb (0.3–30 nm) in MEM + 5% FCS at room temperature, in a volume of 1 ml for 1 h on a rotamix wheel. After 3× 1-ml washes in MEM + 5% FCS, the cells were resuspended in 190 μl of MEM + 5% FCS, and the fluorescence signal derived from europium cryptate-labeled c-Myc mAbs was quantified in a 96-well low fluorescence microplate on the Packard discovery fluorescence plate reader, following the addition of 10 μl of 1 m KF (final concentration, 50 mm KF). In preliminary experiments the inclusion of 5% FCS was found to improve the viability of the cells, and KF was included to stabilize europium cryptate and prevent oxidation of mAbs were in excess nm) to each and the plate was a transfer was the receptor been incubated with for 1 and the signal was for to h of the α1-, β2-, and γ2-subunits was to a c-Myc epitope tag at the and cells expressing c-Myc-tagged α-, β-, or γ-subunits or receptors were by GABAA receptors were measured using two [3H]muscimol, a radioligand for the GABA binding and [3H]Ro15-1788, a radioligand for the benzodiazepine binding which only to fully assembled receptors that an α-, β-, and γ-subunit (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar, D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1150) Google Scholar). In preliminary experiments it was to confirm that a homogeneous of fully assembled receptors was being expressed in the cells. This was in two the number of binding for and [3H]Ro15-1788 was the of two binding and one [3H]Ro15-1788 binding site in a GABA receptor in receptors immunoprecipitated from (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar, K. Ragan C.I. Whiting P.J. McKernan R.M. Mol. Pharmacol. 1994; Google Scholar). if the of receptors is assembled in cell the ratio of the for binding be It is that receptors be expressed that are composed of α- and β-subunits but these would not bind [3H]Ro15-1788, and the ratio would therefore be of and [3H]Ro15-1788 binding was carried out in cells expressing the α1-, β2-, and binding of was of protein with a of binding of [3H]Ro15-1788 was of protein with a of nm The ratio of the was which is in with the expression of αβγ receptors and no significant expression of receptors that only the of antibodies the α1-, β2-, and γ2-subunit to the [3H]Ro15-1788 binding from a solubilized cell was The of receptors be immunoprecipitated with antibodies the α1-, β-, or γ2-subunits β, and of [3H]Ro15-1788 binding This that the receptors that bind [3H]Ro15-1788 at least one α-, one β-, and one The number of c-Myc-tagged subunits was quantified by with anti-c-Myc mAb derivatized with europium cryptate, as in 1 the subunit proposed by N. K. J. Neurosci. 1997; PubMed Google Scholar) as a The of a c-Myc tag on the of the GABAAreceptor subunits not the expression of the The number of binding present on cells was by the addition of the epitope and of [3H]Ro15-1788 binding that there was no major in the of receptors for ligand is as α1β2γ2 α1(c-Myc)β2γ2 α1β2γ2(c-Myc) The density of receptors was by the expression of subunits with a c-Myc epitope being of This is in with studies where GABAA receptors were tagged with c-Myc and or of the GABAA receptor (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). experiments were carried out to determine the for the binding of EuK-labeled c-Myc shown in the fluorescence signal by EuK c-Myc at 620 nm was with receptor the binding of EuK-labeled c-Myc to the receptor was to at least 100 fmol of fluorescence from the plate and other for the fluorescence in the absence of and this was as shown in A. Binding of EuK c-Myc to cells expressing receptors was significant were to to this binding by following with mAb and of cells with to binding on the cells. of these nonspecific binding to cells by more and were not each a type receptors expressed c-Myc epitope and nonspecific binding to these cells was from the total fluorescence which was analogous to the employed for nonspecific binding in a radioligand binding binding experiments were carried out using cells expressing α1β2γ2 and cells expressing receptors where either the α-, β-, or γ-subunit was epitope-tagged with Nonspecific binding of EuK c-Myc to cells expressing an receptor was from total binding at each concentration of antibody and was 50% at nm antibody. In binding was and a and EuK-labeled c-Myc bound to more in the α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2 compared with α1β2γ2(c-Myc) as in To the was on six using nm and the fluorescence counts are shown in 4 A. The ratio of fluorescence to α1β2(c-Myc)γ2, and α1β2γ2(c-Myc) was compared as shown in 4 B. This that there are twice as EuK c-Myc on receptors where the α- or is tagged compared with receptors where the γ-subunit is tagged of signal observed at 620 and 665 nm from cells expressing α1β2(c-Myc)γ2, and shown are the of six experiments. The fluorescence counts observed at 620 nm for α1(c-Myc)β2γ2 were and for α1β2(c-Myc)γ2 were were that observed with α1β2γ2(c-Myc), The and the ratio for binding to α1β2γ2(c-Myc) were for each and are shown in B. The of binding for α1(c-Myc) and were not from in the fluorescence energy transfer where was measured at 665 there was no significant between the fluorescence counts produced by and α1β2(c-Myc)γ2 but were more which was not more A of the for [3H]Ro15-1788 and fluorescence can be used to the number of binding for the binding site. α1(c-Myc)β2γ2 there are fluorescence of binding using nm antibody and 1.8 + this is equivalent to fluorescence of binding that there are 418 fluorescence counts of of antibody there are to be antibody binding binding site. The and of in this of of antibody would be more to to an of the number of antibody binding therefore the observation that there is more one antibody binding binding site the most stoichiometry of benzodiazepine site. The of the c-Myc antibodies for the GABAA subunits was nm as shown in A concentration of nm was for the fluorescence transfer studies because this would half-maximally the with nm c-Myc mAb, cells were washed and incubated with excess c-Myc mAb, and fluorescence at 665 nm was measured in to laser shown in 4 a signal is indicating energy transfer when either the or the β2-subunit is epitope-tagged and but not when the γ2-subunit is Because energy transfer can only when there are two c-Myc in close two subunits in the same receptor this that the stoichiometry of the GABAA receptor under is (α1)2(β2)2(γ2)1. the of signal with α1β2γ2(c-Myc) be as that receptors are not expressed as on the cell surface. this were the it be to energy transfer between γ2-subunits on To date, the stoichiometry of the GABAA receptor has been and Many have observed that a receptor can two of M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar, H. Seeburg P.H. J. 1991; Scopus Google Scholar, J. 1991; PubMed Scopus Google Scholar, S. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.M. Quirk K. Ragan C.I. Whiting 1991; Full Text PDF PubMed Scopus Google Scholar). has been obtained on two of γ-subunit can in a single receptor with two studies that two γ-subunits can be present A. A.L. J. Neurochem. 1994; PubMed Scopus Google Scholar, K. Ragan C.I. Whiting P.J. McKernan R.M. Mol. Pharmacol. 1994; Google Scholar). have been studies on the of two β-subunits because the of the β-subunits has the of which between them. have been studies that the stoichiometry of the The first have an M. U. L. H. J.A. 5: PubMed Scopus Google Scholar) the 2× α, 1× β, and 2× based on measuring the by of amino acids on either of the S. J. Neurosci. 1996; PubMed Google Scholar) used a method of a in to the of the receptor to GABA in to the number of subunits In to M. U. L. H. J.A. 5: PubMed Scopus Google Scholar), conclude that the stoichiometry is 2× α, 2× β, and 1× γ, as do J.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) from studies of subunit The most used antibody of subunits to determine the ratio of subunits present from and conclude that the is also 2× α, 2× β, and 1× N. K. J. Neurosci. 1997; PubMed Google Scholar). The here has used fluorescently derivatized mAbs to quantify subunits to the benzodiazepine binding site on the receptor and fluorescent resonance energy transfer to confirm that the stoichiometry of the receptor is This method has other previously described It intact therefore only receptors that are expressed on the surface are therefore to be are (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, T.P. J. Neurosci. PubMed Google Scholar). It of receptor by only epitope which not the expression or of the receptor (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). small of transiently transfected are this has general applicability to other multisubunit cell surface proteins and can be used to investigate the stoichiometry of subunits in composed of subunits and in other ligand-gated ion We and Mathis of Cis-Bio for for to the assay and for the transfected cells.