Figure 5. Overexpression of GPR158 protein.

TBM-1 cells were transfected at 70–80% confluence using Lipofectamine LTX reagent with either indicated GPR158 expression plasmids or vector alone (A, B and C). Western blotting for the detection of GPR158 in whole cell lysates isolated from cells transfected with indicated plasmids using anti-C-terminal GPR158 antibody (A and B) and anti-GFP antibody (B). Total of 50 µg protein was subjected to SDS-PAGE analysis after boiling with 2-ME as indicated in Materials and Methods. The same membrane was striped and reprobed for β-actin for loading control. (C) The membrane pellet following cytosolic extract isolation from cells transfected with indicated plasmids was boiled in SDS-PAGE loading dye and loaded onto the gel. The western blotting was performed with anti-N-terminus GPR158 antibody. (D) Normal prostate tissue lysates from mice were isolated according to the procedure described in Materials and Methods. The total lysates (30 µg) was subjected to western blotting using either anti-C-terminal GPR158 or anti-N-terminal GPR158 antibodies. The results are representative of three different transfection experiments. ***P<.001; **P<.01; *P<.05; ns, P>.05. (E) The illustration of the location of two cleavage sites and corresponding sizes of the fragments, which are detected using anti-C- and anti-N-terminal GPR158 antibodies. Arrows indicated cleavage sites, I and II, and arrowhead indicate N-glycosylation in N-terminal extracellular domain.