Figure 3.

Immunoblot analysis of TGN38 variants expressed in transfected cell lines. (A) Induction of expression. TGN38 constructs were stably expressed in Cos-7 African green monkey kidney fibroblasts and induced to comparable expression levels from the human metallothionein IIa promoter by the addition of empirically determined concentrations of cadmium chloride (CdCl₂) to the tissue culture medium. Equal numbers of cells were lysed for each sample, and total TGN38 was immunoprecipitated with an excess of sheep polyclonal antiTGN38 (shG29). Immunoprecipitated proteins were detected by immunoblot with a rabbit polyclonal antibody to TGN38 (1918) followed by HRP-conjugated anti-rabbit Ig and ECL. M, mature forms; I, immature forms. (B) Cycloheximide treatment to identify biosynthetic intermediates. Normal rat kidney cells were incubated in the absence or presence of 15 μg/ml cycloheximide for 3 h. Endogenous TGN38 was then immunoprecipitated and immunoblotted as described above; a similar effect of cycloheximide on the immature doublet was observed for all of the transfected cell lines. (C) Comparison of the relative abundance of immature forms of TGN38. The percentage of total TGN38 variant present as an immature lower M_r doublet was determined for each stably transfected cell line by density profile analysis of immunoblots prepared as described above. (±SD, n = 3).