Total lats2

Cells and tissue samples were homogenized using NP40 lysis buffer (FNN0021, ThermoFisher) supplemented with protease and phosphatase inhibitors to obtain total protein extractions. Nuclear and cytoplasmic protein fractions were obtained using the Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher Scientific). Protein concentration in each sample was determined by the bicinchoninic acid (BCA) method with the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Proteins were separated on 7% or 10% gels by SDS-PAGE, transferred onto a nitrocellulose membrane (Amersham Protran GE Healthcare Life Science), and probed with the following primary antibodies: CASR (ab19347, Abcam), phosphorylated YAP (Ser127) (#13008), YAP (#14074), phosphorylated LATS1 (Thr1079) (#8654), total LATS1 (#3477), and total LATS2 (#5888), all from Cell Signaling Technologies. GAPDH (ab9485) and Histone H3 (ab1791), both from Abcam, were used as loading controls for cytoplasmic and nuclear proteins, respectively. The binding of appropriate HRP-conjugated secondary antibodies was revealed using the chemiluminescence ChemiDoc XRS System (Bio-Rad). Analyses of bands densitometries were performed using Image Lab software (Bio-Rad), and protein expression levels were normalized using GAPDH or H3 as reference.

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).