Ab185696

The HaCat cells cultured with or without naringin were harvested and total cell protein was extracted using whole cell lysis buffer. The protein concentrations were determined by the Bradford method (Bio-Rad, CA, USA). Samples with an equal amount of protein were subjected to 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) (Millipore, Bedford, MA, USA) membrane. The membrane was incubated at room temperature in blocking solution (5% nonfat milk) for 1 h followed by incubation for 2 h in blocking solution containing an appropriate dilution of anti-MMP2 (ab86607, abcam), MMP-9 (ab76003, abcam), MMP-14 (ab51074, abcam), TIMP-1 (MAB3300, millipore), TIMP-2 (ab180630, abcam), VEGF-A (ab46154, abcam), VEGF-B(ab185696, abcam), VEGF-C (ab191274, abcam), VEGF-D (ab155288, abcam), VEGF-R1(ab32152, abcam), VEGF-R2 (9698, cell Signaling), VEGF-R3 (2485, cell Signaling), and β actin (E-AB-20058, Elabscience). After washing, blots were then probed with appropriate secondary horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and detected by an ECL detection system (Millipore). β-actin served as internal control.