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. Author manuscript; available in PMC: 2012 Jun 1.
Published in final edited form as: Mol Cell Neurosci. 2010 Oct 30;47(2):71–78. doi: 10.1016/j.mcn.2010.10.002

Figure 2. Cx26V5 co-immunoprecipitates dominant Cx26 mutants.

Figure 2

(A) HeLa cells were transiently transfected to express WT Cx26, Cx26V5, or both Cx26V5 and WT Cx26, as well as WT Cx43, Cx26V5, or both WT Cx43 and Cx26V5. Lysates were immunoprecipitated (IP) with a mouse monoclonal antibody against V5 (MαV5), probed with a rabbit antiserum against the C-terminus of Cx26 (RbαCx26-C, lanes 1–3) or Cx43 (RbαCx43, lanes 4–8), then reprobed with a rabbit antiserum against V5 (RbαV5). Note that the MαV5 co-immunoprecipitated Cx26 (lane 3), but did not co-immunoprecipitate Cx43 (lane 6), which is present in the unbound fraction (UB) of the lysate (lane 8). Because the blot was not stripped before reprobing, faint signals for Cx26 (arrowhead) and Cx43 (triple arrowhead) are present.

(B) HeLa cells were transfected to co-express Cx26V5 and one of the indicated Cx26 mutants. Lysates were immunoprecipitated with MαV5, probed with RbαCx26-C (upper panel), reprobed with RbαV5 (middle panel), then stripped and reprobed again with a rabbit antiserum against the cytoplasmic loop of Cx26 (RbαCx26-L, lower panel). Note that MαV5 co-immunoprecipitates Cx26 mutants in all cases, as shown by the bands in the upper panel and by the lower set of bands in the lower panel. The untagged Cx26 (single arrowhead) ran slightly further in the gel than did the Cx26V5 (double arrowheads). Size markers (in kDa) are shown.