Tris buffered saline with tween 20

WT KG1a, scrambled, or CD34 siRNA knockdown cells were lysed using a cell lysis buffer containing 88% NP40 (Invitrogen™ Novex™, Fisher Scientific), 10% protease inhibitor (Pierce™, Thermo Scientific), 1% PMSF, and 1% Phosphatase inhibitor (Halt™, Thermo Scientific) at 4 °C for 1 h. The whole-cell lysate was collected and incubated with NuPAGE LDS sample buffer (Invitrogen) and 10% β-mercaptoethanol at 70 °C for 10 min. The samples were then run on an SDS-PAGE gel prior to being transferred to a PVDF membrane. The PVDF membrane was blocked overnight at 4 °C using Tris-buffered saline with Tween-20 (Cell Signaling Technology) containing 5% non-fat skim milk powder. The membrane was then washed and incubated with a mouse anti-human CD34 antibody (QBEND/10, BIO RAD). The membrane was then washed and immunoblotted with HRP-conjugated secondary antibodies prior to imaging.

Cell pellets were homogenized and lysed in T-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) with the Halt^TM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) at a ratio of 100:1. The Pierce BCA Protein Assay Kit (ThermoFisher Scientific) was used to determine the protein concentration. Protein denaturation was done in sample buffer (Laemmli Sample buffer: β-ME = 19:1) at 95 °C for 5 min. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was placed onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked in EveryBlot Blocking Buffer (Bio-Rad Laboratories) at room temperature for 15 min and subsequently incubated with primary antibodies (Table 1) at 4 °C overnight. The next day, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Table 1) at room temperature for 1 h. After washing with Tris Buffered Saline with Tween^® 20 (Cell Signaling Technology, Inc., Cambridge, UK), membranes were developed with the Clarity Western ECL Substrate (Bio-Rad Laboratories). Image acquisition was performed on a ChemiDoc^TM MP Imaging System (Bio-Rad Laboratories).

After being washed with ice-cold PBS three times, RA and HD SMSCs were lysed in 0.06 mL of cell lysis buffer (Beyotime) supplemented with a cocktail of protease inhibitors on ice for 30 min. The cells were centrifuged at 14,000 ×g for 30 min at 4°C, and then supernatants were collected. A BCA Protein Assay kit (CWBiotech) was used to measure the protein concentrations. After all the samples were boiled with 20% sample loading buffer (Beyotime), 20 μL of each protein extract was electrophoresed using 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The PVDF membranes were blocked in Tris-buffered saline with Tween-20 (Cell Signaling Technology) and 5% nonfat milk for 60 min at room temperature and then incubated overnight at 4°C with primary antibodies against GAPDH, p53, p-JNK, p-ERK, and p-p38 (dilution 1 : 1000; Cell Signaling Technology). The PVDF membranes were incubated with appropriate secondary antibodies (dilution 1 : 3000; Santa Cruz) for 60 min at room temperature. Then, protein expression levels were detected using enhanced chemiluminescence (Millipore) and quantified using ImageJ software (National Institutes of Health, USA).