Phosphor fak

Total protein samples were extracted by lysing cells with an equivalent Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Haimen, China). The total cellular protein samples were separated by electrophoresis on SDS-PAGE gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Biorad). The membrane was blocked with 5% nonfat milk or BSA in TBST buffer at room temperature for 2 h, and incubated with primary antibodies raised against phosphor-Erk, total-Erk, phosphor-FAK, total-FAK, and GAPDH (Cell Signaling Technology, Inc., Boston, MA, USA) at 4 °C, with sufficient contact overnight. Then the membrane was incubated with secondary antibodies. The blots were measured and visualized by enhanced chemiluminescence (ECL) reagent, followed by ImageJ software quantification and normalization.

The A7r5 cell line was bought from ATCC (Manassas, VA, USA). Recombinant human CTGF/CCN2 was purchased from R&D Systems (Minneapolis, MN, USA). Angiotensin II was purchased from Sigma-Aldrich (St. Louis, MO, USA). Kahweol was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies were purchased from the following vendors: Akt, Erk, FAK, phosphor-Akt, phosphor-Erk, and phosphor-FAK from Cell Signaling Technology (Danvers, MA, USA), CTGF, fibronectin, and YAP from Santa Cruz Biotechnology (Dallas, TX, USA), SM22α and α-SMA from Abcam (Cambridge, MA, USA), and β-actin from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from RMBIO (Missolula, MT, USA). Penicillin/streptomycin antibiotics and trypsin-EDTA solution were purchased from Hyclone (South Logan, UT, USA).

Antibodies against MITF (12590; Cell Signaling Technology, Danvers, MA, USA), GAPDH (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-21 (Santa Cruz Biotechnology; sc-6246), CDK-2 (Cell Signaling Technology; 2546), CDK1 (Santa Cruz Biotechnology; sc-136014), cyclin A (Santa Cruz Biotechnology; sc-271682), cyclin B1 (Santa Cruz Biotechnology; sc-245), cyclin E (Santa Cruz Biotechnology; sc-377100), phosphor-FAK (Cell Signaling Technology; 3283), FAK (Cell Signaling Technology; 3285), F-actin (ab205; Abcam, Cambridge, UK), phospho-YAP (Cell Signaling Technology; 4911), YAP (Cell Signaling Technology; 4912), lamin B (Santa Cruz Biotechnology; sc-6216), Rac1–3 (Cell Signaling Technology; 2465), Cdc42 (Cell Signaling Technology; 2466), phospho-Rac1/Cdc42 (Cell Signaling Technology; 2461), RhoA (Cell Signaling Technology; 2117 or Santa Cruz Biotechnology; sc-418) and CRIK (Santa Cruz Biotechnology; sc-390437) were used in the Western blot and immunofluorescence analyses. CN03 was purchased from Cytoskeleton (Denver, CO, USA).