Bca protein determination kit

All oligonucleotides used in this research Supplementary Table S1 were synthesized by Sangon Biotech. Co., Ltd. (Shanghai, China) with HPLC purification. Peptide was synthesized and purified by Shanghai Scipeptide Co., Ltd. (Shanghai). Other chemical reagents were all of analytical grade. All solutions were prepared with Milli-Q water (18.2 MΩ cm⁻¹) purified with a Milli-Q purification system (Millipore, USA). Nile Red, Z-DEVD-FMK and lysozyme were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD. Caspase-1, caspase-3 and trypsin were purchased from Abcam Trading Co., Ltd. (Shanghai). Bovine serum albumin (BSA) and BCA protein determination kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. Commercial caspase-3 Activity Detection Kit was purchased from Beyotime Biotechnology. Ultrafiltration centrifuge tubes were purchased from Millipore (Shanghai) Trading Co., Ltd.

RIPA tissue lysis buffer, PMSF, protease inhibitor, and phosphoprotease inhibitor were added to the rabbit myocardial tissue at the ratio of 100:1:1:1, respectively, and tissue lysis was carried out on ice. The total protein concentration was quantified by a BCA protein determination kit (Solarbio, Beijing, China). Briefly, equal amounts of proteins (30 μg) were subjected to SDS-PAGE and then transferred onto the PVDF membranes. The membranes were blocked with 5% skim milk and incubated with primary antibodies (collagen Ibs-10423R 1: 800, collagen IIIbs-0549R 1: 800, α-SMAbs-0189R 1: 500, CTGFbs-22361R 1: 1,000, TGF-β1+2 + 3 bs-4538R 1: 1,000, Smad2bs-0718R 1:1,000, Smad39531 1:800, Smad7SC-1001152 1:800, MMP2bs-4605R 1:1,500, MMP9bs-4593R1:1,000, TIMP1bs-0415R 1:1,000, and β-actin 66009-1-Ig 1:5,000 Bioss, Beijing, China) overnight at 4°C. The membranes were washed and then incubated with horseradish peroxidase–labeled secondary antibody at room temperature for 1 h. The immunoreactive bands were visualized using ImageJ (version 1.8.0), and β-actin was adopted as a loading control.

The IL-1β, TNF-α, IL-6, IL-22, IL-17A, or ICAM-1 levels were measured by commercial kits purchased from MULTI SCIENCES (Hangzhou, China). In brief, the psoriatic tissues were homogenized and centrifuged, the supernatant of HaCaT cells was collected, then the protein concentrations were respectively determined by the BCA protein determination kit (Solarbio, Beijing, China). The proteins were diluted to 1 mg/ml, next, the standard curve was established as per the user’s manuals, then the OD value was detected with a microplate spectrophotometer (ELx-800, Biotek Instruments, VT, USA).