Anti lats1

Total proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China), and electrophoresed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked in Quick Block (Beyotime, Shanghai, China) for 15 min, then incubated with specific primary antibodies (1:1000) at 4°C overnight. Anti-PAX5 (Biorbyt, Cambridge, UK), anti-slug, anti-E-cadherin, anti-vimentin, anti-p-LATS1, anti-LATS1, anti-p-YAP, and anti-YAP (Cell Signaling Technology, USA) were used. Membranes were incubated in secondary antibodies at room temperature for 1 hour. TBST was used to wash unbound antibodies. An enhanced chemiluminescence detection system was used to detect target proteins. GAPDH and β-actin were internal controls.

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).

Anti-MST1 (Cell Signaling Technology, USA), anti-LATS1 (Cell Signaling Technology, USA), anti-YAP (Cell Signaling Technology, USA), anti-YAP (Santa Cruz, USA), anti-PP2A Bβ (Proteintech, China), anti-Myc (Proteintech, China), anti-FLAG (Proteintech, China), or anti-IgG (Santa Cruz, USA) antibodies were incubated with Protein A/G Magnetic Beads (Bimake, USA) for 4 h at 4°C. Samples were lysed with Thermo Scientific Pierce IP Lysis Buffer (Thermo Fisher Scientific, USA) with protease inhibitor mixture (Bimake, USA) and incubated with an antibody-bead complex overnight at 4°C. The immunoprecipitation products were then precipitated by the antibody-bead complex using a magnetic rack and analyzed by western blotting.