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.

(A) 对异位表达空载体（Vector）或血凝素标记HBx（HA-HBx）的Huh7细胞提取物，采用抗CDH1抗体进行蛋白质印迹检测，并以GAPDH作为内参完成归一化处理。(B) 采用实时定量逆转录PCR（qRT-PCR），以文件S1中表S1所列引物，检测空载体或HA-HBx转染细胞中CDH1的相对mRNA水平；以GAPDH作为内参。数据（柱状图）以三次独立重复实验的平均值±标准差（mean ± SD）表示。(C) 对仅表达空载体、共表达空载体与HA-CDH1，以及共表达HA-CDH1与HA-X0的细胞裂解液，采用指定抗体分别对USP37、CDC6、Geminin及GAPDH进行蛋白质印迹检测。(D) 对未处理组，以及经CDK2抑制剂II化合物3（10 µM处理6小时）处理的空载体或HBx转染细胞提取物，分别对USP37、CDC6及GAPDH进行蛋白质印迹检测。采用USP37抗体对未处理或经CDK2抑制剂II化合物3（10 µM处理6小时）的空载体或HBx转染细胞的裂解液开展免疫沉淀实验，以磷酸化丝氨酸抗体及USP37抗体对免疫复合物进行蛋白质印迹检测。同时对总细胞裂解液采用磷酸化CDC6（Ser-54）抗体及GAPDH抗体完成蛋白质印迹检测。(E) 对表达空载体或HA-HBx的Huh7细胞提取物，采用抗β-TrCP抗体进行蛋白质印迹检测，并以GAPDH作为内参完成归一化处理。(F) 采用实时定量逆转录PCR（qRT-PCR），以文件S1中表S1所列引物，检测空载体或HA-HBx转染的Huh7细胞中β-TrCP的相对mRNA水平；以GAPDH作为内参。数据（柱状图）以三次独立重复实验的平均值±标准差表示，#代表p<0.001，具有统计学显著性差异。(G) 对转染Myc-β-TrCP、Myc-ΔF-β-TrCP及HA-HBx（按分组指示）的Huh7细胞进行裂解，将裂解液经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分离后，采用对应抗体对USP37、β-连环蛋白（β-catenin）及CDH1进行蛋白质印迹检测；以GAPDH作为上样对照。