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. 2019 Feb 14;116(10):4661–4670. doi: 10.1073/pnas.1813244116

Fig. 7.

Fig. 7.

Rack1 stabilizes HDAC1/HDAC2 protein levels by inhibiting their polyubiquitination. (A and B) Representative Western blots and quantification show a dose-dependent increase in HDAC1/HDAC2 expression in HEK293 cells transfected with exogenous Flag-tagged Rack1. MG132-treated cells were detected as a positive control (**P < 0.001, n = 3). IB, immunoblot; MW, molecular weight. (C and D) Representative Western blots and quantification show a time-dependent increase in HDAC1 expression in HEK293 cells transfected with exogenous Flag-tagged Rack1 (**P < 0.001, n = 3). (E and F) Expression vectors encoding Flag-tagged HDAC1 (Flag-HDAC1) and hemagglutinin-tagged ubiquitin (HA-Ub) were transfected either with or without GFP-tagged Rack1 (Rack1-GFP) into HEK293 cells in the presence of 20 μg/mL MG132 to inhibit proteasome-dependent degradation. Protein extracts were immunoprecipitated with either anti-Flag or anti-HA and immunoblotted for HA or Flag, respectively. IgG was used as a negative control. The black arrowhead indicates Flag-HDAC1 (n = 4). IP, immunoprecipitate. (G and H) HEK293 cells were cotransfected with Flag-HDAC1 and HA-MDM2 with or without Rack1-GFP, and protein extracts were immunoprecipitated with anti-HA or anti-Flag and immunoblotted for Flag or HA, respectively. IgG was used as a negative control. The black arrowhead indicates Flag-HDAC1 (n = 4).