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Fig. 2. Western blot analysis of wild-type and mutant Cx43-eYFP fusion protein expression and quantitation of surface expression. Stable C6R cell lines expressing wild-type and indicated mutant Cx43-eYFP constructs were generated using the retroviral BH-RCAS expression vector. (A) Equal amounts (20 µg) of lysates prepared from uninfected control cells (C6R) or representative clones of C6R cells stably expressing wild-type Cx43-eYFP (wild type) or eGFP (eGFP) were probed with an anti-GFP polyclonal antibody. A 
70 kDa band representing the full-length fusion protein was produced by wild-type cells but not C6R or eGFP cells. There was also a faint 30 kDa band detected from wild-type lysates that co-migrates with the eGFP band and represents either an independently transcribed species or a degradation product. (B) Lysates prepared from C6 cells, uninfected control C6R cells (C6R) or C6R wild-type Cx43-eYFP (WT) or mutant Cx43-eYFP constructs were also probed with an anti-Cx43 polyclonal antibody. The full-length fusion protein (70 kDa band) is again detected for wild-type cells, as well as each of the mutants tested but not detected in C6 or C6R cells. All cell lines produce a 40 kDa band that represents endogenous Cx43. Probing the same blot for actin demonstrates that equal amounts of lysates were loaded. (C,D) Surface proteins were selectively biotinylated at 4°C and isolated with streptavidin-agarose beads. Biotinylated samples were recovered from roughly 12-fold more lysate than was loaded in A. The full-length wild-type Cx43-eYFP 70 kDa band was again detected with both antibodies. No band was seen after recovery of biotinylated proteins from the eGFP lysate after probing for GFP indicating that non-specific biotinylation of cytoplasmic proteins did not occur (C). Actin bands were also not detected in the surface fraction (D). (E) Quantitation of cell-surface wild-type or mutant Cx43-eYFP fusion protein was performed by measuring eYFP fluorescence on a fluorescence microplate reader prior to loading on gel. Data shown were obtained from one to four experiments on two independent clonal cell lines for wild type and for each mutant. Relative fluorescence units/µg protein from total lysates are given.
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