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. Author manuscript; available in PMC: 2017 May 5.
Published in final edited form as: Acta Histochem. 2012 Jan 12;114(7):705–712. doi: 10.1016/j.acthis.2011.12.006

Fig. 2.

Fig. 2

Biochemical evidence for human TMEM176A and 176B protein-protein interaction and subcellular co-localization of heterologously expressed human TMEM176A and 176B proteins in HEK-293 cells. (a) Representative Western blot image showing the antibody specificity of anti-HsTMEM176A (left panel, green text) and anti-HsTMEM176B (right panel, orange text) polyclonal antibodies (pAbs). Expression constructs were transfected into HEK-293 cells, and the cells were lysed 24 h post-transfection. Left panel lanes 1: negative control (untransfected) lysate; 2: over-expressed HsTMEM176A lysate; 3: co-expressed HsTMEM176A and 176B lysate; 4: overexpressed HsTMEM176B lysate (control); 5: eluted HsTMEM176A from co-IP sample; 6: total protein input from co-IP sample. Right panel lanes 1: negative control (untransfected) lysate; 2: over-expressed HsTMEM176A lysate (control); 3: co-expressed HsTMEM176A and 176B lysate; 4: over-expressed HsTMEM176B lysate; 5: eluted HsTMEM176B from co-IP sample; 6: total protein input from co-IP sample. (b) Amino acid sequence alignment of HsTMEM176A and HsTMEM176B proteins using Clustal W software analysis tool. The amino acid epitopes for anti-HsTMEM176A and anti-HsTMEM176B are underlined (green and orange lines, respectively). (c) Representative Western blot image of HsTMEM176A-HA (single transfection control; left panel) and HsTMEM176A-HA co-expressed with HsTMEM176B-Myc/DDK (right panel). The samples were co-immunoprecipitated with anti-DDK mAb, while eluted samples were detected with anti-HA pAb as described in the Materials and Methods section. Lanes: I, input of total protein lysate; W, wash; E, elution. Green arrowheads indicate HsTMEM176A (monomer or tetramer); blue arrowhead represents mouse immunoglobulin (IgG). (d) Representative confocal micrographs of HsTMEM176A-GFP (left panel), HsTMEM176B-mCherry (middle panel) and composite image stained with DAPI (right panel). DAPI stains the nuclei blue. Both TMEM proteins co-localize along the plasma membrane (arrows), and appear as punctate, vesicle-like structures within the cytoplasm. Note that TMEM176B did not always co-localize in these structures (arrowheads). Scale bar = 10 μm. Hs, Homo sapiens; WB, Western blot; IP, immunoprecipitation; mAb, monoclonal antibody; pAb, polyclonal antibody; 1-mer, monomer; 4-mer, tetramer; IgG, immunoglobulin G.