E ab 20058

Cells were lysed with RIPA buffer (50 mM Tris–HCl at pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA plus proteases and phosphatases inhibitors) for 1 h at 4 °C. Total protein quantification was performed with Bio-Rad Protein Assay Dye Reagent concentrate. A total of 40 μg of protein was separated on 15% SDS–PAGE gels at 100 V and transferred to PVDF membranes for 2 h at 200 mA. PVDF membranes were hybridized with antibodies against LAMC2 (AMAB 91,098, Sigma-Aldrich), SMAD2 (cell signaling – 5339), pSMAD2 (cell signaling – 3108S), GAPDH (cell signaling – 2118), β-actin (E-AB-20058, Elabscience), MMP-2 (4022S, Cell Signaling), Vimentin (3932, Cell Signaling) and α-tubulin (2144, Cell Signaling) and subsequently hybridized with peroxidase-conjugated goat anti-mouse or anti-rabbit Ig secondary antibodies (DPVR-HRP, Immunologic), and then visualized by enhanced chemiluminescence (ECL Nova 2.0 XLS071, 2050 Cyanagen). n $\ge 3.$

Cells were lysed in RIPA buffer (CW2333S) supplemented with protease inhibitor cocktail (CW2200S) and phosphatase inhibitors (CW2383S). Total protein was extracted from tissue samples by a Tissue Protein Extraction Kit (CW0891M) (all from CWBIO, Beijing, China). Then, the protein concentration was determined using a BCA protein assay kit (Beyotime). Total proteins from cells (20 μg) and tissues (20 μg) were separated by SDS PAGE through a 10% gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010, MA, USA) at 100 V for 60 min. After blocked with Tris-buffered saline containing Tween-20 (TBST, 1000 : 1) and 5% fat-free milk for 2 h, the membranes were incubated at 4°C overnight with primary antibodies against SCD1 (1 : 1000; Bioss, Cat# bs-3787R, Beijing, China), p-PI3K (1 : 1000; Cat#: 17366; Cell Signaling Technology, Danvers, MA, USA), PI3K (1 : 1000; clone: 1F6A7; Proteintech, Wuhan, China), phospho-AKT (Ser473; 1 : 1000; Cat# 4069; Cell Signaling Technology, Danvers, MA, USA), AKT (1 : 1000; Cat# 4691; Cell Signaling Technology), and actin (rabbit polyclonal; 1 : 2000; Cat# E-AB-20058; Elabscience, Wuhan, China).

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.