Fluorchem fc system

The cells were lysed in RIPA buffer (Heart Biological Technology Co., Ltd., Xi'an, China) containing 1% aprotinin, 1% activated Na₃VO₄ and 1% PMSF on ice. The protein concentration was detected according to the instructions of the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Total proteins (50 µg) were loaded onto a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, separated by electrophoresis and transfered onto PVDF membranes (Millipore, Boston, MA, USA). The membranes were blocked with 5% skim milk at room temperature for 2 h and then incubated with rabbit anti-human PKCζ (1:1,000, #sc-216; Santa Cruz Biotechnology, Inc., CA, USA), rabbit anti-human MMP2 (1:1,000, #10373-2-AP), MMP9 (1:500, #10375-2-AP), mouse anti-human β-actin (1:1,000, #60008-1-lg) (both from China Branch of Proteintech Co., Hubei, China) and rabbit anti-human phospho-PKCζ antibodies (1:500, #9378; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight. The following day, the membranes were sequentially incubated with HRP-conjugated goat anti-rabbit IgG (1:3,000, #A0208) or HRP-conjugated goat anti-mouse IgG (1:3,000, #A0216) (Beyotime, Shanghai, China) at 37°C for 1 h. Immunoreactive proteins were detected using ECL reagent (Millipore) and the FluorChem FC system (Alpha Innotech, San Leandro, CA, USA). Bands were quantitated using ImageJ software.

Total protein lysates were generated using RIPA lysis buffer supplemented with protease and phosphatase inhibitor mixtures (KC-440; Shanghai KangChen Biological Technology Co., Shanghai, China). Nuclear protein extracts were obtained using the NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer's instructions. Proteins (40 µg) were loaded onto a 5–10% polyacrylamide gel, separated by electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% non-fat milk, the PVDF membrane was incubated with rabbit polyclonal antibodies to collagen I (ab96723) and III (ab7778) (both from Abcam, Cambridge, MA, USA), p-Smad2 (3108), Smad2 (3122), p-Smad3 (9520), Smad3 (9523) (all from Cell Signaling Technology, Inc.), HIF-1α (ab51608; Abcam) and histone H3 (sc-8654-R; Santa Cruz Biotechnology) or mouse polyclonal α-SMA antibody (BM0002; Wuhan Boster Biological Technology, Ltd., Wuhan, China) and β-actin antibody (4970; Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated goat anti-rabbit (BA1054) or anti-mouse (BA1050) antibody (Wuhan Boster Biological Technology, Ltd.) was used as a secondary antibody. Proteins were visualized by enhanced chemiluminescence system using FluorChem FC system (Alpha Innotech, San Leandro, CA, USA).

To extract proteins from cells and tissues, fibroblasts and scar tissue were collected, washed twice with PBS and lysed in cell lysis buffer (RIPA, Beyotime) supplemented with protease inhibitors (PMSF, China Bost). After lysis the samples were incubated on ice for 15 min. Cell debris was then removed by centrifugation at 12,000 x g for 5 min at 4°C. The protein concentration was determined using a BCA kit (Beyotime). Total protein (50 μg)_was run on a 10% SDS-PAGE gel and transferred to a PVDF transfer membrane (Millipore, Billerica) at 100 V for 40–100 min. The membrane was then blocked with 5% skim milk for 1 h at room temperature in TBST and probed with the following antibodies: COL1 (1 : 1000, Abcam, Cambridge, UK), COL3 (1 : 1000, Abcam, Cambridge, UK), α-SMA (1 : 1000, CST, USA), CD9, CD63 (1 : 1000, Proteintech, China), Smad2 antibody sampling kit (1 : 1000, CST) and β-actin (1 : 1000, Xi’an Johnson & Johnson) and incubated overnight at 4°C. The next day, the samples were incubated with enzyme-labeled anti-rabbit IgG secondary antibody (1 : 3000, Bots, Wuhan, China) for 1 h at 37°C. Proteins were detected by chemiluminescence with an ECL kit (ECL Kit, MA, USA), immunoblotting of membranes was probed with a FluorChem FC system (Alpha Innotech), protein expression intensity was analyzed with ImageJ software and β-actin was used to normalize.