Figure 6.

Caveolin-1 exhibits direct protein–protein interaction with Oct4. A, H460 cell lysates were prepared and immunoprecipitated (IP) with anti-Oct4 antibody or control IgG and then analyzed Cav-1 and phosphorylated Cav-1 (Tyr-14) by Western blotting assay. B, H460 cell lysates were prepared and immunoprecipitated with anti-Cav-1 antibody or control IgG and then analyzed Oct4 with Western blotting assay. C, H460 cell lysates were prepared and immunoprecipitated with anti-phosphorylated Oct4 (Ser-236) antibody or control IgG and then analyzed the Cav-1 expression by Western blotting assay. D, H460 cell lysates were prepared and immunoprecipitated with anti-phosphorylated Cav-1(Tyr-14) antibody or control IgG, then evaluated Oct4 protein level by Western blotting. In all experiments, immunoblots were performed on cell lysates used as input for immunoprecipitation (IP) using GAPDH antibody to confirm equal loading of the samples. E, protein–protein interaction of Cav-1 and Oct4 was confirmed by PLA in H460 cells. After plating H460 cells for 24 h, the cells were permeabilized and stained with rabbit anti-Cav-1 and mouse anti-Oct4 for 24 h. The stained cells were subjected to PLA following the manufacturer's instruction. The interaction between Cav-1 and Oct4 was visualized by fluorescence microscopy (×20). Cells were stained with Hoechst 33342 dye to aid visualization of the cell nucleus. The Cav-1:Oct4 complexes appear as green fluorescence.