Fig. 3

SIRT7 deacetylates CHD1L to stabilize its proteins. A Tandem affinity purification of CHD1L-containing protein complex was conducted using 786-O cells stably expressing double tag Flag-HA-CHD1L. Mass spectrometry was used to analyze the peptides. B Co-immunoprecipitation (Co-IP) of endogenous CHD1L with anti-SIRT7 antibodies (upper) and endogenous SIRT7 with anti-CHD1L antibodies (lower) in 786-O cells. Western blot was used to show the results. C Western blotting assay showing the CHD1L proteins (upper) and mRNA (lower) in cells transfected with increasing Myc-SIRT7 plasmids. D, E Western blotting assay detected the SIRT7 expressions in control and SIRT7-KD cells. F Western blot of CHD1L proteins in WCLs of 786-O or Caki-1 cells transfected with the indicated SIRT7 plasmids for 48 h and then treated with CHX (50 μg/ml) and harvested at different time points. G Acetylation level of CHDL1 in 786-O cells treated with shCtrl or shSIRT7 lentiviruses probed with pan anti acetyl lysine antibodies. Lower is quantitative data showing an increased level of CHDL1 acetylation in cells with SIRT7 KD. H Acetylation levels of CHDL1 in control or SIRT7-OE cells. Lower is quantitative data showing an increased level of CHDL1 acetylation. I Flag-CHD1L, Myc-SIRT7, and HA-Ub were co-transfected into 786-O cells. Immunoblots with anti-HA antibody showing polyubiquitination of CHD1L in the presence or absence of ectopic SIRT7 or H187Y mutant. Cells were treated with MG132 (15 µM) for 12 h. J Immunoblots with anti-HA antibody showing polyubiquitination of CHD1L in control or SIRT7-KD cells. Cells were treated with MG132 (15 µM) for 12 h. K Immunoblots showing CHD1L proteins in cells transfected with increasing doses of Myc-SIRT7 or H187Y plasmids. The CHD1L mRNA levels in the indicated groups were shown on the right. *p < 0.05, **p < 0.01, ***p < 0.001, ns no significant