Fig. 3.

The shift of S1P and S2P to ER is associated with the monomerization of GCC185. (A) Representative 3D SIM imaging of LNCaP cells: control and GCC185 KD. Cells were co-stained with S1P (green, left panel) or S2P (green, right panel) and calreticulin (red); bars, 2 μm. White boxes indicate the area magnified below. (B) Representative 3D SIM imaging of PC-3 cells co-stained with S1P or S2P (green) and GCC185 or calreticulin (red); bars, 2 μm. (C, D) Quantification of the Pearson coefficient of colocalization for the indicated proteins in cells from A and B, respectively (N = 10 cells for each series of SD SIM imaging; **P < 0.01, t test). (E, F) Golgi fractions isolated from LNCaP and PC-3 cells were subjected to sucrose sedimentation analysis on a 5–25% sucrose gradient. 5% (E) and 25% (F) fractions were collected and analyzed by 4–15% gradient SDS-PAGE and probed with GCC185 Ab. The samples were prepared under low (1%) concentrations of β-mercaptoethanol, and the same amount of proteins were loaded. (G) AFM topography images of GCC185 from PC-3 and LNCaP cells; bar, 500 nm. (H and I) Statistical histogram of GCC185 protein volume distribution for PC-3 (H, n = 424) and LNCaP (I, n = 194) samples