Phosphatase inhibitor

PDLSCs were washed with ice‐cold PBS and lysed using a RIPA lysis buffer containing 1% PMSF (Solarbio) and 1% phosphatase inhibitor (Boster, Wuhan, China) and then were centrifuged at 12,000 g for 10 min. Protein concentration was measured by a BCA Protein Assay Kit. Proteins were added with SDS‐PAGE loading buffer and then denaturated at 100°C for 5 min. 20 μg/lane proteins were loaded to SDS‐PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with non‐fat dry milk for 1 hr, blotted primary antibodies overnight at 4°C and subsequently incubated with horseradish peroxidase‐labelled secondary antibodies (1:2000; Beyotime, Shanghai, China) for 1 hr at room temperature. The membrane was washed three times in tris‐buffered saline with 1‰ Tween20 (TBST). The immunoreactive bands were visualized using enhanced chemiluminescence reagents (Millipore). The level of each protein was normalized to GAPDH before statistical analysis. Image J 1.44 software (NIH, Bethesda, Maryland, USA) was used to quantify the protein expression. The primary antibodies used were as follows: rabbit anti‐phospho‐ERK1/2 (1:1000; Cell Signaling Technology, Danvers MA, USA), rabbit anti‐ERK1/2 (1:2000; Cell Signaling Technology), rabbit anti‐phospho‐p38 (1:1000; Cell Signaling Technology) and GAPDH (1:10,000; Proteintech, Chicago, IN, USA).

For the analysis of Bcl2, bax, LC3B, osteosarcoma cells were resuspended in a protein lysis buffer RIPA (Boster bio, Wuhan, China) containing protease inhibitors (Boster Bio, Wuhan, China) and phosphatase inhibitor (Boster Bio, Wuhan, China) at 4°C for 30 min incubation. After homogenization, the supernatants containing protein was collected by centrifugation and stored at –80°C. Followed by the BCA protein assay, protein samples were separated by 10%–12% SDS-PAGE electrophoretically and were then transferred to a PVDF membrane (GE Healthcare life Sciences, Piscataway, USA). Then western blotting were proceeded with primary and secondary antibodies and was detected with peroxidase-conjugated anti-rabbit antibody followed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, USA). The details of the primary antibodies and dilution factors were as follows: Bcl2 (Cell Signaling Technology, Inc. USA, 1:1000), bax (Cell Signaling Technology, Inc. USA, 1:1000), LC3B (Cell Signaling Technology, Inc. USA, 1:1000), GAPDH (Cell Signaling Technology, Inc. USA, 1:1500).

Skin tissue was homogenized in RIPA (Radio Immuno Precipitation Assay) lysis buffer, protease inhibitor, and phosphatase inhibitor (Boster, Wuhan, China) to extract the total protein. The protein concentration was determined using BCA protein assay kit. The protein was denatured in gel sample buffer at 100 °C for 5 min, separated by 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (EpiZyme, Shanghai, China), and blocked with 5% (w/v) non-fat dry milk in TBST buffer (Tris-Buffered Saline with Tween-20). The membrane was incubated with the primary antibody against NF-κВ (BA1297-2, Boster, Wuhan, China), TNF-α (BA14901, Boster), IL-6 (BA4339-2, Boster), COX-2 (BA3708, Boster), ERK (GB112238, Servicebio, Wuhan, China), JNK (M02608-3, Boster), P38 (A00176-2, Boster), AP-1 (GB11270, Servicebio), MMP-1 (A00733-1, Boster), and MMP-2 (A00286, Boster) at 4 °C overnight, followed by washing at TBST, and then incubated with goat anti-rabbit HRP (horseradish peroxidase)-IgG (BA1055, Boster) as the secondary antibody for 45 min. After washed with TBST 3 times for 10 min, membrane was incubated in SuperSignal ECL Western substrate (Biosharp, Hefei, China). The protein band was visualized using the Chemiluminescent Imaging System (Tanon-5200SF, Shanghai, China). The intensity was quantified by Image J version 1.8.0 program.