Total mst1

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).

RNA was isolated using the TRIzol reagent (Invitrogen) and tested for integrity by denaturing gel electrophoresis. Primers were designed with Applied Biosystems software, and PCR was performed using SYBR Green according to the “standard curve” method. Results were normalized to actin and GAPDH to control for mRNA abundance.
For Western blots, cells were lysed in SDS sample buffer, separated by SDS-PAGE, transferred to nitrocellulose, and serially probed with antibodies to Nore1 (Thermo Fisher Scientific), total Mst1 (Cell Signaling Technology), phospho-Mst (Cell Signaling Technology), or GAPDH (loading control). Each experiment was repeated with at least three different lysates, and a representative blot is shown.