Figure 3. HBx differentially regulate CDH1 and β-TrCP to circumvent USP37 downregulation.

(A) Cell extracts of Huh7 cells ectopically expressing Vector or HA-HBx were western blotted with anti-CDH1 antibody and normalized with GAPDH. (B) Relative m-RNA levels of CDH1 in Vector or HA-HBx transfected cells, were measured by performing qRT-PCR with primers mentioned in Table S1 in File S1. GAPDH was used as control. Data (bar diagrams) are shown as mean ± SD of three independent observations. (C) Cell lysates from cells expressing vector alone; co-expressing vector and HA-CDH1 and HA-CDH1 and HA-X0 were western blotted for USP37, CDC6, Geminin and GAPDH with indicated antibodies. (D) Untreated and CDK2 Inhibitor II compound 3 (10 µM for 6 h) treated Vector or HBx transfected cell extract were western blotted for USP37, CDC6 and GAPDH. Immunoprecipitation assay was performed using USP37 antibody with cell lysates from untreated or CDK2 inhibitor II compound 3 (10 µM for 6 h) treated Vector or HBx transfected cells. The immune-complexes were blotted with phospho-serine and USP37 antibody. Total cell lysates were also western blotted with phospho-CDC6 (Ser-54) and GAPDH antibody. (E) Cell lysates from cells expressing Vector or HA-HBx were western blotted with anti-β-TrCP antibody and normalized with GAPDH. (F) Relative m-RNA levels of β-TrCP in Vector or HA-HBx transfected Huh7 cells, were measured by performing qRT-PCR with primers mentioned in Table S1 in File S1. GAPDH was used as control. Data (bar diagrams) are shown as mean ± SD of three independent observations # represents statistically significant difference of p<0.001. (G) Myc-β-TrCP, Myc-ΔF-β-TrCP and HA-HBx transfected huh7 cells (as indicated) were lysed and lysates was separated on SDS-PAGE gel western blotted for USP37, β-catenin and CDH1. GAPDH was used as a loading control.