Tween 20 tbst

Total proteins were extracted in cell dialysis buffer containing protease inhibitors and quantified using a BCA Protein Assay kit. The proteins were denatured, separated (40 µg protein/lane) by electrophoresis equipped with 10% SDS-PAGE and transferred to a PVDF membrane (200 mA, 110 min). The membranes were blocked with Tris-buffered saline (Well-Biology Co., Ltd., Changsha, China) with Tween-20 (TBST; Beyotime Institute of Biotechnology) containing 5% skimmed milk at 37°C for 1 h. The membranes were incubated overnight at 4°C with primary antibody diluted at the following appropriate concentrations: MMP8 (1:400), MMP13 (1:400), MMP14 (1:400), MMP17 (1:400), TIMP1 (1:400), TGF1 (1:400), Beclin 1 (1:1,000), Atg3 (1:1,000), Atg7 (1:1,000), PI3K (1:1,000) and AKT1 (1:1,000). Following washing with TBST buffer three times, the membranes were incubated with secondary antibody (1:8,000) for 1 h at 37°C. Stripes were visualized using an enhanced chemiluminescence detection reagent (Wuhan Boster Biological Technology, Ltd., Wuhan, China) and analyzed using Alpha Imager 2200 (ProteinSimple, San Jose, CA, USA). GAPDH served as the internal reference.

Transfected cells were lysed in radio-immunoprecipitation assay (RIPA, Beyotime Biotechnology, Shanghai, China) buffer with 1% phenylmethylsulfonyl fluoride (PMSF) to acquire the total cellular proteins, and protein concentration was evaluated with a G-250 Bradford kit (Beyotime Biotechnology). To detect the expression of FLAG-WT-PHEX and FLAG-MUT-PHEX in the transfected HEK-293T cells, the soluble proteins were then separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane, where FLAG-PHEX ran at a size of approximately 180 kDa. The membrane was blocked in 5% milk, incubated with primary FLAG monoantibody (1:5000; Abmart, Shanghai, China), and diluted in 5% bovine serum albumin (BSA, CWBIO Technology Co. Ltd, Beijing, China) overnight at 4°C. The membrane was then washed with tris-buffered saline using Tween 20 (TBST; Beyotime Biotechnology) buffer three times, each for 15 minutes. Afterwards, the membrane was incubated with secondary horseradish peroxidase (HRP)-conjugated antibody (antimouse, 1:5000; CWBIO Technology Co. Ltd) diluted in 5% milk. Enhanced chemiluminescence (ECL) mix (Merck Millipore, Darmstadt, Germany) was used to visualize proteins on radiographs.

The proteins (2.6 µg/µl) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and western blotting was subsequently performed, as previously described (11 (link)). Briefly, the cells were harvested, resuspended in cell lysis buffer (Beyotime Institute of Biotechnology) and incubated on ice for 30 min. The cell lysates were centrifuged at 12,000 x g for 10 min at 4°C. The supernatants were mixed with one-quarter volumes of 4X SDS sample buffer and boiled for 10 min. The proteins were subsequently separated by SDS-PAGE in a 10–15% gel. Following electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk powder in Tris-buffered saline (Thermo Fisher Scientific, Inc.) with Tween-20 (TBST; Beyotime Institute of Biotechnology) for 1 h at room temperature. The membrane was subsequently incubated with a diluted primary antibody in blocking buffer overnight at 4°C. The membrane was washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (Beyotime Institute of Biotechnology) for 30 min at room temperature. Following extensive washing, the proteins were visualized using an ECL-Plus kit and the blots were exposed to Kodak radiographic film (Kodak. Corp., Rochester, NY, USA).