Fig. 1.

HAUSP stabilizes Rb. (A) GFP-HAUSP was overexpressed in HEK293 cells and Rb, p107 and p130 protein levels were determined by WB. Lamin B serves as the loading control (HAUSP, upper band in lane 2 indicates GFP-tagged HAUSP; Rb, two bands may represent differentially phosphorylated forms; p130, lowermost band at 130 kDa represents the specific form). (B) GFP-HAUSP was overexpressed in COS-7 cells and Rb protein levels were determined by WB. Actin serves as the loading control. (C) Rb transcript levels were quantified by quantitative real-time PCR (qRT-PCR) upon HAUSP overexpression. Data represent the mean ± SEM from three biological repeats in triplicate; P = 0.96 (ns). (D) WB was done to verify the effect of siRNA-mediated partial knockdown of HAUSP on Rb protein expression using specific siRNAs for 72 h. (E) WB was performed to verify the effect of shRNA-mediated HAUSP partial knockdown on Rb protein expression. Two rounds of transfections were performed and cells were harvested after 72 h. (F) WB was performed to verify HAUSP knockdown and the effect on Rb protein expression using two different shRNAs. The values have been estimated by densitometry and normalized against the loading control actin (left panel). Graphical representation of HAUSP and Rb expression levels in HAUSP knockdown conditions from two independent repeats; P = 0.0073 (**), 0.0226 (*), 0.9 (ns) and 0.022 (*) (right panel). (G) Under similar conditions of HAUSP knockdown, Rb transcripts were quantified by qRT-PCR. Data represent mean ± SEM from three biological repeats in triplicate; P = 0.56 (ns). (H) For the rescue experiment, specific siRNA was used to knockdown HAUSP and the protein level of Rb was estimated by WB (left panel). Under similar conditions HAUSP was exogenously overexpressed and Rb level was estimated by WB (middle panel). The values have been estimated by densitometry and normalized against the loading control actin. Densitometric analysis of protein levels from three repeats of the rescue experiment show significant decrease in Rb level with knockdown of HAUSP [P = 0.004 (*)] while overexpression of HAUSP leads to rescue of Rb levels [P = 0.057 (ns)] (right panel). (I) Cells were transfected either with empty vector (left panel) or GFP-HAUSP (right panel) and treated with CHX (50 μg·mL⁻¹) for a CHX chase assay to estimate the Rb protein levels in the presence or absence of HAUSP for the indicated time points in hours. (J) Graph showing Rb half-life in the absence or presence of HAUSP. Data represent mean ± SD from three independent experiments. ns, not significant; *, significant; **, very significant; ***, extremely significant.