Anti sav1

Antibodies against caspase 9 (sc-56077) and β-actin (sc-8432) were obtained from Santa Cruz Biotechnology, Texas, United States. Others were purchased from Cell Signaling Technology: anti-PARP (#9542), anti-cleaved caspase 3 (#9661), anti-MST1 (#3682), anti-MST2 (#3952), anti-p-MST1(T183)/MST2(T180) (#49332), anti-LATS1 (#3477), anti-p-LATS1(S909) (#9159), anti-p-LATS1(T1079) (#8654), anti-MOB1 (#13730), anti-p-MOB1 (T35) (#8699), anti-SAV1 (#13301), anti-YAP (#14074), anti-p-YAP (S127) (#13008), and anti-p-YAP (S397) (#13619). RNase A and Propidium Iodide/PI were purchased from Coolaber (Beijing, China) and Sigma-Aldrich (United States), respectively.

Cellular or tissue samples were lysed with RIPA lysis buffer freshly adding cocktail protease inhibitor (Thermo Scientific). Equal amounts of cellular proteins were subjected to electrophoresis in SDS-PAGE, and transferred to nitrocellulose membranes (Millipore). The membranes were blocked and then incubated at 4 °C overnight with indicated first antibodies, followed by incubation with the appropriate HRP-conjugated secondary antibodies (Thermo Scientific). The protein bands were detected and analyzed with an ECL. The following antibodies were used: anti-Aurora A (Upstate, #07-648), anti-phospho-Aurora A (Thr288) (Cell Signaling Technology, #3079), anti-YAP (Cell Signaling Technology, #4912), anti-phospho-YAP(ser127) (Cell Signaling Technology, #4911), anti-phospho-YAP(Ser397) (Cell Signaling Technology, #13619), anti-Lats1 (Cell Signaling Technology, #3477 P), anti-phospho-Lats1 (Thr1079) (Cell Signaling Technology, #9654P), anti-Lats2 (Rui Ying biological, China, #RLP1047), anti-SAV1 (Cell Signaling Technology, #3507P), anti-MST1 (Cell Signaling Technology, #3682P), anti-GAPDH (Kangcheng, China,#KC-5G4), anti-Lamin B1 (Epitomics, #6581-1), anti-CTGF (Life Science Products & Services, #AB60212a), anti-Myc Tag (MERCK, 05-724).

The transfected cells were lysed by RIPA (Solarbio, Shanghai, China) for total protein extracts, which were quantified by BCA method. Then, the extracts were separated on 10% SDS-PAGE gel. According to sandwich structure, the electrotransfer was assembled to move the protein into PVDF membranes (GE Healthcare, Piscataway, NJ, USA), which were further incubated with the primary antibodies as follow: anti-MST1, anti-SAV1, anti-LATS1, anti-YAP1 and anti-GAPDH antibody (Cell Signaling Technology). The membranes were incubated with second antibody and measured by ECL kit (ECL Amersham).