Figure 3.

Immunofluorescence localization of PS-2 proteins in HeLa cells. (A–D) HeLa cells transiently transfected with PS-2+ Myc and stained for PS-2 expression using antibodies specific for either the PS-2 NH₂ terminus (A and C) or the myc tag at the COOH terminus (B and D). The anti–PS-2 antibody was preincubated with either purified GST nonfusion protein for A and B or GST–NH₂-terminal fusion protein for C and D to demonstrate specificity of the anti–PS-2 NH₂-terminal antibody. Double staining of the transfected cells with the preincubated PS-2 and anti-myc antibodies demonstrate that staining is abolished only upon incubation with GST fusion proteins containing NH₂-terminal PS-2 sequences. Staining for both NH₂- and COOH-terminal epitopes showed complete colocalization (compare A and B), suggesting the two epitopes are not dissociated (not proteolytically cleaved and differentially localized). (E–J) HeLa cells transiently transfected with constructs pPS-2+Myc (E and F), pPS-2(166aa)+Myc (G and H), and pPS-2(138aa)+Myc (I and J) and double stained for PS-2 and calreticulin. The left and right panels show the same cells double-stained for either PS-2 expression, using the anti– NH₂-terminal PS-2 antibody (E, G, and I), or for endogenous calreticulin (F, H, and J). Please note complete colocalization of PS staining with calreticulin in pPS-2+Myc– and pPS-2(166aa)+Myc– expressing cells but not with pPS-2(138aa)+Myc–transfected cells. Bar, 10 μm.