Figure 7

SDIM1 physically interacts with DNAJB4. A. Interaction between SDIM1 and DNAJB4 in yeast two-hybrid system. Empty or SDIM1 containing pGBKT7 bait vector and empty or DNAJB4 containing pACT2 library vector were co-transformed into yeast host cells AH109 and plated onto SD/-Trp -Leu -Ade -His + X-α-gal plate. a - a negative test of empty bait vector and DNAJB4, b - a negative test of SDIM1 bait plus empty library vector, c - a positive test showing the interaction between SDIM1 and DNAJB4, d - a standard positive control showing interaction between p53 and T antigen. B. Total cellular proteins were extracted from HEK-293 cells co-transfected with flag-tagged DNAJB4 and EGFP-tagged SDIM1 constructs and immunoprecipitated with anti-flag antibody. The presence of DNAJB4 in the complex was detected by anti-flag antibody. Lane1 - total cellular proteins extracted from untransfected cells, lane 2 - total cellular proteins extracted from cells co-transfected with pCMV-DNAJB4-Tag1 and pSDIM1-EGFP, lane 3 - mock immunoprecipitation without primary anti-flag antibody, lane 4 - proteins immunoprecipitated with anti-flag antibody. C. The presence of SDIM1 in the complex, shown in B, was revealed by Western blotting with anti-SDIM1 antibody. Lane assignment is the same as those in B. D. Co-localization of SDIM1 and DNAJB4. Mouse primary neurons were fixed and stained with anti-SDIM1 anti-DNAJB4 antibodies. FITC-conjugated anti-rabbit IgG (for SDIM1) and rhodomine-conjugated anti-mouse IgG (DNAJB4) were used to detect the specific immunostaining. The nuclei were stained with DAPI and viewed with a Zeiss Axiovert 200 M fluorescence microscope. a. DAPI stained nuclei. b. Anti-SDIM1 staining. c. Anti-DNAJB4 staining. d. a, b and c overlay. Arrowheads indicate an apoptotic cell. Scale bar in a = 50 μM.