Fig. 2. Association of endogenous Stat3 and GRIM-19. (A) Evaluation of the GRIM-19 antibody. COS-1 cells were transfected with GST vector (lanes 1, 3 and 5) or GST–GRIM-19 (lanes 2, 4 and 6). Cells were lysed and subjected to western blot analysis using pre-immune serum (PI, lanes 1 and 2), purified GRIM-19 antibody (α-G-19; lanes 3 and 4) or GRIM-19 antibody pre-absorbed by bacterially produced GRIM-19 (lanes 5 and 6). (B) Western blot analysis of endogenous GRIM-19 in MCF-7 cells induced by IFN-β (500 U/ml) and RA (1 µM) for various times. The blot was re-probed with anti-β-actin as a control. (C) Association of endogenous Stat3 and GRIM-19 in various cell types. MCF-7 cells were left untreated (lanes 1 and 2) or treated with IFN-β and RA for 48 h (lane 3). C2C12, SH-SY5Y and PC12 cells were grown under normal conditions. Cell lysates were incubated with either pre-immune serum (PI) or anti-GRIM-19 (α-G-19) as labeled on top of the figures, and blotted with anti-Stat3 (upper panels). The blots were stripped and re-probed with anti-GRIM-19 (lower panels).