Bca assay kit

Total proteins were isolated from plasma samples and cells using RIPA reagent (Sigma‐Aldrich, USA), and the protein concentrations were determined by a Bicinchoninic Acid Protein (BCA) Assay Kit (Abcam, Shanghai, China). 20 μg proteins were separated by 10% SDS‐PAGE (Thermo Fisher Scientific Inc, Waltham, MA, USA) and transferred to a polyvinylidene difluoride (PVDF; Sigma‐Aldrich, USA) membrane. The membrane was then blocked by 5% non‐fat milk for 1 h at about 25°C and incubated with primary antibodies: rabbit anti‐SRGN (1:1000, ab156991; Abcam, Shanghai, China) and β‐actin (1:1000, ab8227; Abcam, Shanghai, China) at 4°C overnight. Afterward, the membrane was washed with PBS (0.1% Tween‐20) three times and then incubated with IgG H&L (HRP) secondary antibody (1:1000, ab7090; Abcam, Shanghai, China) at room temperature for 2 h. β‐actin functioned as an internal control. ImageJ version 1.46 software (National Institutes of Health, Bethesda, MD, USA) was applied to quantify the band intensity, and relative SRGN protein expression was normalized to the band intensity of β‐actin.

Protein was extracted from cells on ice with precooled RIPA lysis buffer (contains protease inhibitors; Beyotime Biotechnology) for 10 min, and then quantified using a BCA assay kit (Abcam, Shanghai, China). Protein samples (30 µg/lane) were separated by 10% SDS-PAGE at 120 V and transferred to PVDF membranes (Millipore, Thermo Fisher Scientific) at 280 mV for 90 min. The membranes were first blocked with 5% skimmed milk for 2 h at room temperature. Afterward, they were incubated with diluted primary antibodies against CTRP6 (ab36898; 1:500), PPARγ (ab178860; 1:1,000), Bcl2 (ab32124; 1:1,000), Bax (ab32503; 1:1,000), Cleaved caspase3 (ab32042; 1:500) and GAPDH (ab181602; 1:10,000) at 4°C overnight followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (ab6721; 1:10,000; all Abcam) at room temperature for 1 h. The chemiluminescence reaction was performed using an ECL kit (Yeasen BioTechnology, Shanghai, China), and densitometry was analyzed using the ImageJ software (v1.8; National Institutes of Health) [23 (link)].

The protein of each group was collected after cell lysis with RIPA buffer and quantified using a BCA assay kit from Abcam (Cambridge, MA, USA). 20 µg of protein was separated on 10% SDS-PAGE gel, electrotransferred to nitrocellulose membranes (Thermo Fisher, USA), blocked with 1% BSA for 1 h and incubated with rabbit primary antibodies in 1:1000 dilution, including anti-Ang-2(#2948S), anti-FOXC2(#12974S),anti-E-cadherin(#14472S), anti-vimentin(#5741S), anti-fibronectin(#4705S) and anti-β-actin(#4970S) (Cell Signaling Technologies,USA). Subsequently, the membranes were washed extensively with PBST (Tween-20, 0.1%) followed by incubation of secondary antibody, HRP-conjugated anti-rabbit IgG incubation for 1 h at room temperature. The ECL method was then used to visualize protein bands.

Fibroblasts were lysed using pre-chilled lysis buffer at 4℃. The BCA assay kit (Abcam, USA) was employed to quantify the total protein content in each sample. Measured aliquots of 30 µg total protein per sample were subjected to electrophoresis on a 4-20% gradient SDS-PAGE gel and transferred onto PVDF membranes. Membranes were blocked using a solution of 5% skim milk in TBS-T and incubated with primary antibodies, all from Abcam, against KLF5 (1:200), Runt-related transcription factor 2 (RUNX2, 1:1000), osteocalcin (OCN, 1:1000), osteopontin (OPN, 1:1000), CX43 (1:1000), and β-actin (1:200) at 4℃ overnight. Following primary antibody incubation, membranes were treated with peroxidase-conjugated anti-rabbit IgG secondary antibody (1:5000, Millipore Corporation). Enhanced chemiluminescence reagent (Merck Millipore, Billerica, MA, USA) and a chemiluminescence imaging system (Bio-Rad Laboratories, Inc) were used for visualization. Protein bands were quantified using ImageJ software version 1.52 (National Institutes of Health, USA), with β-actin as the loading control.