Figure 2. HBx upregulates USP37 mRNA and protein levels.

(A) Cell lysates from Huh7 cells transfected with Vector control or HA-X0 construct were run on SDS-PAGE gel and were blotted for USP37 and normalized against GAPDH (B) Total RNA was isolated and USP37 mRNA levels were measured by RT-qPCR using specific primers (Table S1 in File S1) in Huh7 cells overexpressing Vector, HA-HBx, E2F1 and E2F1-1-374-ΔC as indicated. Data (bar diagrams) are shown as mean ± SD of three independent observations # represents statistically significant difference of p<0.001. (C)Stability of USP37 protein was monitored in Huh7 cells transfected with vector or HA-HBx then treated with 20 µg/ml cycloheximide for the indicated durations. Change in Endogenous USP37 protein levels were detected by western blotting using antiUSP37 antibody as indicated in line graph. GAPDH was used as a control. Data (line graph) are shown as mean ± SD of three independent observations. (D) Cell extracts from Huh7 cells transfected with Vector or HA-X0 as indicated, were treated with 20 µM of MG132 for 6 hrs and western blotted for USP37 protein and normalized with GAPDH. (E) Ubiquitination assay was performed by immunoprecipitating USP37 from cell lysates from Huh7 cells transiently transfected with Vector, HA-HBx, X-E and Myc-Ubiquitin as indicated (1 ug vector, 2 ug scrambled shRNA and 2 ug Myc-ubiquitin; 1 ug HA-HBx, 2 ug scrambled shRNA and 2 ug Myc-ubiquitin or 1 ug HA-HBx, 2 ug X-E shRNA and 2 ug Myc-ubiquitin were transfected in 100 mm dishes) and treated with MG132, as mentioned above and western blotting the immino-complexes with anti-Ubiquitin Anitibody.