Figure 2.

Antibodies that did not recognize rat α7 nAChR expression in GH4C1 cells. Representative images of the western blot and immunofluorescence results for four different α7 nAChR (α7) antibodies are shown. Left panel: Western blot data showed no ostensible difference between WT and α7 or α7-GFP transfected cells (Lane 1: GH4C1-WT, Lane 2: GH4C1 eGFPN1, Lane 3: GH4C1 α7-GFP, Lane 4: GH4C1 α7). Right panel: Immunofluorescence results demonstrate identical staining pattern between untransfected and α7 transfected GH4C1 cells. Strong nonspecific binding can be seen with ab23832 and sc-5544 α7 nAChR antibodies. Abcam ab23832 (lot GR89867-1) antibody used at a 1:1000 dilution for both immunoblotting and immunofluorescence showing no significant difference between WT and α7-transfected cells. 1:1000 dilution of sc-58607 (lot L0312) does not detect any significant bands in GH4C1 cells but a 1:100 dilution of the same antibody detects multiple bands throughout the blot in different molecular weight region irrespective of α7 presence. Only mild immunofluorescence staining was seen in the GH4C1 cells with sc-58607 antibody at a 1:1000 dilution. Sc-5544 (lot D2214) showing intense immunoblot and immunofluorescence staining at a 1:1000 dilution in both WT and α7-transfected GH4C1 cells. EMD Millipore MABN529 (lot Q2459834) antibody showing no immunofluorescence staining at a 1:1000 dilution but showing nonspecific binding on the western blot. Scale bar, 40 μm. Abbreviations: nAChR, nicotinic acetylcholine receptor; WT, wild-type; GFP, green fluorescent protein.