Fig. 2

GCC185 is the Golgi binding partner for S1P and S2P. (A) S1P and S2P W-B of the cis-medial-Golgi and trans-Golgi fractions (0.6 M/0.8 M and 0.25 M/0.6 M sucrose interface, respectively); samples were normalized by the total protein concentration. Giantin and TGN46 were used as loading controls for cis-medial- and trans-Golgi, respectively. (B) IF staining of LNCaP cells (left panel) and PC-3 cells (right panel) to estimate colocalization of S1P and S2P with giantin and GCC185; bars, 10 μm. (C) Quantification of the Pearson coefficient of colocalization for the cells presented in B (N = 90 cells from three repeats; **P < 0.001, *P < 0.01, t test). (D) S1P and S2P W-B of the GCC185 immunoprecipitation (IP) sample prepared from LNCaP cells. (E) GCC185 W-B of the lysate or the protein complex pulled down from the lysate of LNCaP cells using biotinylated hS1P or hS2P full-length peptides and Dynabeads M-280 Streptavidin. (F) GCC185 W-B of the lysate of RWPE-1, LNCaP, and PC-3 cells; β-actin is a loading control. (G, H) GCC185 W-B of the lysate of LNCaP cells treated with scramble or different combinations of GCC185 siRNAs. (I) S1P and S2P W-B of the ER fraction isolated from LNCaP cells: control and GCC185 KD; HSP70 and TGN46 are loading controls for ER and trans-Golgi, respectively. (J) GCC185 W-B of the lysates from LNCaP cells: transfected with control siRNAs and empty pCMV6-AC vector, transfected with GCC185 siRNAs and empty pCMV6-AC vector, and transfected with GCC185 siRNAs followed by GCC185 plasmid corresponding to the WT hGCC185. (K) S1P and S2P W-B of the ER fraction isolated from LNCaP cells presented in J. (L) Representative images of colonies formed by control and GCC185 KD LNCaP cells as described in the Materials and Methods section; bars, 200 μm. (M) Quantification of the colonies’ lengths (the longest diameter) in 30 randomly selected areas for control and GCC185 KD LNCaP cells. Data were collected from three independent experiments and expressed as a mean ± SD; * P < 0.01, t test. (N) ATF6 and AR W-B of the nuclear fraction from control and GCC185 KD LNCaP cells; lamin B1 is a loading control