Ab70565

Cells were harvested and lysed in 0.5 ml lysis buffer plus protease inhibitors (Roche) for 1 h on rotor at 4 °C. After 12,000g centrifugation for 15 min, the lysates were incubated with 2 μg specific antibody overnight at 4 °C, 30 μl protein A/G-agarose beads (Santa Cruz, SC-2003) were washed with lysis buffer and then added for additional 3 h. Thereafter, the beads were washed five times with lysis buffer and boiled with loading buffer for 5 min and subjected to SDS–polyacrylamide gel electrophoresis for analysis. Flag-peptides were used for elution were purchased from ChinaPeptides. The following antibodies were used for immunoprecipitation: antibodies against Zyxin (Epitomics, 3586-1, EPR4302), Flag (Sigma, F1804, clone M2), Myc (Santa Cruz, sc-40, clone 9E10) and Lats2 (Abcam, ab70565).

Western blot assays were performed as described in our previous study [17 (link)]. Briefly, cultured cells or ccRCC tissues were lysed in RIPA lysis buffer, and protein concentrations were determined using the BCA assay kit according to the manufacturer’s protocols (Thermo Fisher Scientific, Waltham, MA, USA). The nuclear and cytoplasmic proteins from ccRCC cells were extracted using an NE-PER Nuclear and Cytoplasmic Extraction Kit (#78899; Thermo Scientific). The primary antibodies used were rabbit anti-RNF43 antibody (ab84125; Abcam), rabbit anti-LATS1 + LATS2 antibody (ab70565), rabbit anti-LATS1 + LATS2 (phospho T1079 + T1041) antibody (ab111344), rabbit anti-YAP antibody (ab52771), rabbit anti-YAP (phospho S127) antibody (ab76252), rabbit anti-GAPDH antibody (#5174), rabbit anti-β-actin antibody (#58169), and rabbit anti-histone H3 antibody (#4499), all of which were from Cell Signaling Technology (Danvers, MA, USA).