FIG. 4.

MC007L interacts with the pRb/E2F-1 complex. (A) MC007L targets pRb and E2F-1 at the mitochondria in transient transfection assays. Saos-2 cells were transiently transfected with MC007L-RFP (first row) or with the MC007L mutant with mutated LxCxE motifs (second row) together with Myc-tagged pRb and E2F-1-expressing plasmids. After 24 h posttransfection, cells were fixed and stained with a MAb specific for Myc and a rabbit anti-E2F-1 antibody. In the third row, Saos-2 cells transfected with MC007L-RFP together with a Myc-tagged pRb 756/757 and E2F-1 plasmids were subjected to immunofluorescence 24 h posttransfection. Cells were fixed and stained with a MAb specific for Myc and a rabbit anti-E2F-1 antibody. Two secondary antibodies were applied with FITC or Cy5, respectively. Bar, 15 μm. (B) MC007L precipitates the endogenous pRb/E2F-1 complex. MC007L coimmunoprecipitates the endogenous pRb/E2F-1 complex. CV1 cells transfected with empty vector, MC007L-FLAG, or MC007L-143/180-FLAG were subjected to coimmunoprecipitation using an anti-FLAG-specific antibody. Precipitates were analyzed by immunoblot analysis using the antibodies indicated below. MC007L was detected using an anti-FLAG antibody and E2F-1 was detected with the MAb KH95. To detect endogenous pRb, the blot was reprobed with the anti-pRb-specific antibody G3-245 and β-actin-specific MAb, respectively. One-tenth of the loading control was used.