Phospho lats1 ser909

Cumulus cells from 30 or 75 COCs or 20 μg of whole ovary lysates from eCG or hCG (6 and 24 h) primed female mice were denatured by boiling for 5 min in Laemmli sample buffer (with 5% 2-Mercaptoethanol), followed by quenching on ice and prepared for immunoblotting as previously described [55 (link)]. Proteins were separated on a 4–12% Bis-tris gel (Novex NuPAGE) and transferred to PVDF membrane (0.2 μm). The membranes were blocked in TBST+ 5% BSA for 1 h with shaking at room temperature, followed by incubation with 1:1000 diluted phospho-LATS1 (Ser 909) (Cell Signaling Technology, 9157), phospho-YAP1 (Serine 127) (Cell Signaling Technology, 13008), phospho-TAZ (Ser 89) (Santa Cruz, 17610), YAP1 (Cell Signaling Technology, 14074), TAZ (Abcam, ab84927) or β-actin (ACTB, 1:6000, Sigma) antibodies with agitation at 4 °C overnight. Following incubation, blots were washed 3–4 times, 10 min each with 1 X TBST, and incubated with HRP-labeled secondary antibody (1:50,000) for 1 h at room temperature in the dark. Blots were washed and Pierce ECL Plus substrate (Life Technologies, 80197) was added for 5 min before detecting signal in a phosphorimager (GE STORM 860) or a Bio-Rad XRS+ gel documentation system.

Cells were harvested followed by lysis and fixed amount of protein was loaded on a polyacrylamide gel followed by transfer to a PVDF membrane, followed by incubation with shaking overnight at 4°C with antibodies against phospho-YAP (Ser-127), total YAP/TAZ, phospho-MST1/2, total MST1, total MST2, phospho-LATS1(Ser-909), total LATS1, total LATS2 (all from Cell Signaling, Beverly, MA), GPR40, GPR120 (Abcam, Cambridge, United Kingdom), GAPDH and YAP (Santa Cruz Biotechnologies, Santa Cruz, CA) diluted in TBS containing 5% milk and 0.1% Tween-20. Signal was detected using the chemiluminescence (ECL) system (Millipore, Darmstadt, Germany).

BNIP‐2 (HPA026843) antibody was purchased from Sigma‐Aldrich. RhoA (sc‐418), Actin (sc‐4778), tubulin (sc‐5286), GAPDH (47724), cTnT (sc‐20025), and Myl2 (sc‐517414) antibodies were purchased from Santa Cruz. MLC2 (#3672), Phospho‐MLC2 Thr18/Ser19) (#3674), MST‐1 (#3682), Phospho‐MST1 (Thr183)/MST2 (Thr180) (#49332), YAP/TAZ (#8418), phospho‐YAP (Ser127) Antibody #4911, Phospho‐TAZ (Ser89) (#59971), Lats1 (#9153), and Phospho‐Lats1 (Ser909) (#9157) were purchased from Cell Signalling Technology. Rabbit IgG (sc‐2027) and mouse IgG (sc‐2025) were from Santa Cruz Biotechnology. HRP secondary antibodies polyclonal antibody against FLAG and polyclonal antibody against HA were from Sigma. All Alexa Fluor dyes and Alexa Fluor Phalloidin were from Life Technologies. For Rho activity assay, Rho inhibitor I (Cat. No. CT04) and Rho activator II (Cat. No. CN03) were from Cytoskeleton, Inc. Dimethyl sulfoxide (DMSO), blebbistatin, and all‐trans retinoic acid were from Sigma‐Aldrich.