Ab150766

After exposure or not to LIPUS, HaCaT cells were lysed in RIPA buffer (Beyotime, China) containing 1 mM phenylmethyl sulfonyl fluoride (PMSF; Beyotime). The whole lysates were centrifuged at 12,000 g for 10 min at 4°C, and the protein levels in the supernatants were quantified using the BCA™ Protein Assay Kit (Pierce, USA). Proteins were loaded onto SDS-PAGE gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Then, PVDF membranes were blocked with 5% bovine serum albumin (BSA; Solarbio, China), and incubated overnight at 4°C with specific primary antibodies for cyclin D1 (ab134175), cyclin-dependent kinase (CDK) 6 (ab151247), CDK4 (ab137675), vascular endothelial growth factor (VEGF; ab150766), matrix metalloproteinase (MMP) 2 (ab97779), MMP-9 (ab137867), PI3K (ab180967), phospho (p)-PI3K (ab182651), β-actin (ab8227, all Abcam), AKT (#9272), p-AKT (#9271), JNK (#9252), and p-JNK (#9251, all Cell Signaling Technology, USA). After rinsing, PVDF membranes were incubated with HRP-conjugated secondary antibody (goat anti-rabbit, ab205718, Abcam) at room temperature. An enhanced chemiluminescence (ECL) kit (Thermo Scientific, USA) was used to detect the target proteins. Protein levels were quantified by ImageJ software (National Institutes of Health, USA).

Total proteins were extracted from cultured cells by RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China) with protease inhibitors (Protease inhibitor cocktail; Roche, Indianapolis, IN). Protein concentration was determined by using BCA protein assay (Thermo Fisher Scientific, U.S.A.). The protein samples (30 μg) were separated by SDS/PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, U.K.). The membranes were blocked with 5% nonfat milk/0.1% Tween 20 in phosphate-buffered saline (PBS-T) for 1 h at room temperature. Then the membranes were incubated with primary antibodies diluted appropriately in PBS-T at 4°C overnight. Antibodies specific to VE-cadherin (ab33168, 1:2000), VEGF (ab150766, 1:2000), β-catenin (ab32572, 1:2000), matrix metalloproteinase (MMP) 2 (MMP2) (ab97779, 1:2000), MMP9 (ab38898, 1:2000) and GAPDH (ab8245, 1:2000) were supplied by Abcam (Cambridge, U.K.). GAPDH was used as a loading control. After the incubation of primary antibodies, the membranes were washed with PBS-T for three times, 5 min each. Then the membranes were incubated with HRP–conjugated secondary antibodies (Cell Signaling Technology, Boston, U.S.A., 1:5000 in 5% nonfat milk/PBS-T) at room temperature for 1 h. After washing, the protein signals were visualized by ECL detection reagent (Amersham Biosciences, Castle Hill, Australia).