Enhanced chemiluminescence system

Cells were lysed in RIPA and protein concentrations were determined using the BCA protein assay kit (P0010, Beyotime Biotechnology, Shanghai, China). Equal amounts of proteins (40 μg) were resolved by 10% SDS-PAGE and the proteins were transferred to Hybond ECL membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used included anti- vimentin, Smad2/3/4, phosphorylated Smad2/3/4, E-Cadherin, cytokeratin, F-actin, Snail/Slug, Twist, Zeb1 and β-actin. After washing with TBST, the membranes were probed with HRP-labeled secondary antibodies. The membranes were visualized using an enhanced chemiluminescence system (Kodak, Rochester, NY, USA).The densitometric data of Western blots of the three independent experiments were combined.

rotein contents were extracted from cells using radio immunoprecipitation lysis buffer (#89901; Thermo Fisher Scientific, Waltham, USA) containing proteinase and phosphatase inhibitors (Thermo Fisher Scientific), and the concentration was determined using a BCA protein assay kit (#P0011; Beyotime, Shanghai, China). After separated by SDS-PAGE, protein samples were transferred to the PVDF membrane (#GVWP02500; Sigma-Aldrich, St. Louis, USA). GAPDH was used as a loading reference. The membrane was subsequently incubated with primary antibodies against TGM2 (#ab2386; 1:1000, Abcam, Cambridge, UK), alkaline phosphatase (ALP; #ab229126; 1:500, Abcam), osteocalcin (OCN; #ab93876; 1:1000, Abcam), and runt-related transcription factor 2 (RUNX2; #ab236639; 1:1000, Abcam) at 4 °C overnight followed by further incubation with secondary antibodies. An enhanced chemiluminescence system (Kodak, Rochester, USA) was applied to visualize the signals on the membrane. ImageJ software (NIH, Bethesda, USA) was used analyze the signal intensity of the blots.

Proteins were extracted from tissue samples using a protein extraction reagent (Sigma, USA), following a protocol provided by the manufacturer. Cells were lysed in radioimmunoprecipitation assay (RIPA; P0013B, Beyotime Biotechnology, Shanghai, China), and protein concentrations were determined using the BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to Hybond ECL membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with primary antibodies including anti-cPLA2, anti-p-cPLA2, anti-Atrogin-1, anti-MuRF-1, and anti-HIF-1α at 4°C overnight. After washing with TBST, the membranes were probed with HRP-labeled secondary antibodies. The membranes were visualized using an enhanced chemiluminescence system (Kodak, Rochester, NY, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Western blots were performed in a standard protocol. Protein concentrations were determined using the BCA protein assay kit. Equal amounts of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to Hybond ECL membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with primary antibodies including anti-JAK1/2, anti-p-JAK1/2, anti-STAT3/5, anti-p-STAT3/5, and anti-LC3B I/II at 4°C overnight. After washing with TBST, the membranes were probed with HRP-labeled secondary antibodies. The membranes were visualized using an enhanced chemiluminescence system (Kodak, Rochester, NY, USA). GAPDH was used as a loading control.

Proteins from cultured neutrophils were prepared with a protein extraction solution (containing 20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 0.1% SDS and 1% protease inhibitor cocktail). Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher, Waltham, MA, USA). Then, 40 μg samples of protein extracts were fractionated on 12% polyacrylamide-sodium dodecyl sulfate (SDS) gels and then transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) fat-free milk in Tris-buffered saline (TBS) containing 0.05% Tween-20, followed by incubation with primary antibodies to phosphorylated-p38 MAPK (p-p38 MAPK), phosphorylated Mcl-1 (p-Mcl-1), Akt, phosphorylated P65 (p-P65), phosphorylated IκBα (p-IκBα), and α-tubulin (all at 1∶1000; Abcam, Cambridge, UK) at 4°C overnight. The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1∶5000, Abcam), and bands were visualized with an enhanced chemiluminescence system with short exposure to X-ray films (Kodak, Rochester, NY, USA).

Total cellular proteins were harvested by incubating the cells in the RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS) obtained from Cell Signaling Technology. The protein concentrations were determined by BCA kit (Sigma-Aldrich) according to the manufacturer's protocols. SDS-PAGE was performed in 10% tricine gels with equal amounts of protein loaded per lane. After electrophoresis, proteins were transferred from the gel to a nitrocellulose membrane at 110 V for 1 hour, and then the membrane was blocked with 5% nonfat milk in TBST buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, and 0.1% Tween-20) for 1 hour. The membranes were then incubated with primary antibodies at 1∶1000 dilution in 5% nonfat milk over night at 4°C, followed by secondary antibodies conjugated with horseradish peroxidase at 1∶2000 dilution for 1 hour at room temperature. Protein bands were visualized on X-ray film using an enhanced chemiluminescence system (Kodak).