Gpr120

The specimen was sequentially fixed in 10% formalin for two days, transferred to ethanol of different concentration and embedded in paraffin in preparation for histopathological analysis. Thin sections (5 μm) were stained with hematoxylin-eosin (H&E) for histopathological study and subjected to immunohistochemical staining of target proteins with the avidin-biotin-peroxidase method. Slides were washed with PBS for five minutes three times and all the experimental steps were taken according to UNIV IHC detection kit operation instruction. Samples were incubated overnight with primary antibody GPR120 (Abcam, Cambridge, United Kingdom) (1:200), GPR40 (GeneTex, Nottingham, United Kingdom) (1:200), YAP/TAZ (Cell Signaling Inc Beverly, MA) (1:200), p-YAP (Cell Signaling, Beverly, MA) (1:200) at 4°C, followed by incubation with HRP-conjugated secondary IgG for 30 min at RT. After washing with PBS three times, 3-3′ diaminobenzidine (DAB) substrate chromogen solution (Envision Plus Kit, Dako Corp) was applied. The reaction was monitored by microscopy and was terminated when properly developed.

Cell lysis was performed using the RIPA (including 1% PMSF) protein extraction solution (Solarbio, Beijing, China). The protein concentrations were measured using the BCA protein concentration assay kit (Solarbio, Beijing, China). SDS–polyacrylamide gel electrophoresis (PAGE) was conducted to separate the extracted proteins with different molecular weights. The target proteins were immediately transferred to nitrocellulose membranes, which were then incubated overnight at 4℃ with antibodies against β-actin (42 kDa; ZSGB-BIO, Beijing, China), β-Tubulin (55 kDa; ZSGB-BIO, Beijing, China), GPR40 (40 kDa; Abcam, Cambridge, USA), GPR120 (40 kDa; Abcam, Cambridge, USA), KLF7 (25 kDa; Abcam, Cambridge, USA), CCL2 (40 kDa; Abcam, Cambridge, USA), CCR2 (35 kDa; San Ying Biotechnology, Wuhan, China), Ki67 (384 kDa; Abcam, Cambridge, USA), and MMP2 (72 kDa; Abcam, Cambridge, USA). Afterward, the membranes were incubated with the secondary antibody at room temperature for 2 h. It needs to be explained that, in order to reduce the mutual interference of polyclonal antibody incubation among multiple antibodies, we have tailored according to MW markers in this study, and each membrane containing different target proteins is incubated separately with corresponding antibodies.The protein bands were detected based on chemiluminescence (ThermoScientific, Waltham, America).

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).