Atxn3

NP-40 lysis buffer supplemented with protease inhibitors (Meilun, China) was used to extract total protein. BCA Reagent (Thermo Scientific, Rockford, IL, USA) was used to measure the protein concentration. We used an SDS–polyacrylamide gel was to separate the total proteins and transferred proteins to 0.45 μm PVDF membrane (Millipore, USA). The primary antibodies used for western blot analysis are listed as follows: YAP (Proteintech, 66,900–1-Ig), ATXN3 (Proteintech, 13,505–1-AP), GAPDH (Proteintech, 60,004–1-Ig), Myc (Proteintech, 60,003–2-Ig), HA (Proteintech, 51,064–2-AP) antibodies. Signals were detected and visualized using ECL (Meilun, China) and ChemiDocMP imager (Bio-Rad).

Total protein was lysed for 30 min in the lysate buffer (Beyotime, Shanghai, China) and washed in pre-cold PBS. The protein was separated by SDS-PAGE, and loaded on PVDF membrane. Blockage of non-specific antigens was conducted in 5% skim milk for 2 h. Membranes were immunoblotted with primary and secondary antibodies for the indicated time points. Bands were finally exposed using Immobilon Western Chemiluminescent HRP Substrate for quantifying the grey value using the ImageQuantLAS 4000 mini biomolecular imager. The following antibodies were used for Western blot analyses: E-cadherin (#3195, CST), N-cadherin (ab18203, Abcam), Snail (#3879, CST), Slug (#9585, CST), Vimentin (#5741, CST), β-actin (AP0733, Bioworld, China), ATXN3 (#13505-1-AP, Proteintech), PI3K (#ab86714, abcam), AKT (#4691, CST), p-AKT (#66444-1-lg, Proteintech).