Glyceraldehyde 3 phosphate dehydrogenase

Proteins extracted from HEC-1-A cells were prepared using RIPA buffer containing the phosphatase inhibitor phenylmethylsulfonyl fluoride (RIPA:phenylmethylsulfonyl fluoride = 100:1). Proteins were quantified with a bicinchoninic acid protein assay kit (Servicebio, Wuhan, China). Then, protein samples were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% gels and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were incubated with primary antibodies against PI3K (1:500 dilution; Proteintech, Chicago, IL, USA), phosphorylated (p)-PI3K (1:1000; BIOSS, Woburn, MA, USA), Akt (1:20000; Proteintech), p-Akt (1:20000; Proteintech), and glyceraldehyde 3-phosphate dehydrogenase (1: 50000; Proteintech). After washing with Tris-buffered saline and Tween 20, PVDF membranes were incubated with the corresponding secondary antibody for 1.5 h at 25°C. An electrochemiluminescence detection reagent (Beyotime Institute of Biotechnology, Shanghai, China) was used to visualize protein bands.

Roswell Park Memorial Institute (RPMI) 1,640 culture medium was purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Olaparib (AZD2281, Ku-0059436) was purchased from Selleck (Houston, TX, USA). ML216 (CID-49852229) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies against pBRCA1 (Ser1524), p53BP1 (Ser1778), pATM (Ser1981), pATR (Ser428), Rad50, Mre11, pDNA-PKcs (Ser2056), Ku80, pChk2 (Thr68), pChk1 (Ser317), pRb (Ser807/811), pRb (Ser795), p21, and γH2AX were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against RPA70, Rad51, 53BP1, CyclinA, and Ki-67 were purchased from Abcam (Cambridge, UK). Antibodies against β-tubulin, β-actin, and glyceraldehyde 3-phosphate dehydrogenase were purchased from Proteintech (Rosemont, IL, USA).

The expression of Apo-1 protein of MSCs was detected by WB analysis. Cells were lysed in RIPA buffer (Beyotime Biotechnology, China). Total protein quantification was performed using the BCA Protein Assay Kit according to the manufacturer's instructions. Then, a capillary-based Simple Western Analysis (ProteinSimple, CT, USA) was used to detect protein abundance according to the manufacturer's protocol. Briefly, 0.6 μg of whole protein sample was loaded into each well, and the size of the separation matrix ranged from 12 to 230 kDa. The target protein was identified using primary antibody-APO1, and glyceraldehyde 3-phosphate dehydrogenase (ProteinTech, IL, USA) was used as a reference control. According to primary antibodies, rabbit secondary antibody provided by the manufacturer was used. Chemiluminescent signals were detected and quantified using Compass Software (ProteinSimple), and the area value was used as the protein expression level.

Total protein was extracted from lung tissues exposed to OVA by lysis solution (mixed with 1% phenylmethylsulfonyl fluoride) and was quantified by using a Bicinchoninic Acid Protein Assay Kit (Solarbio, Beijing, China). 20 μg protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by a transfer to polyvinylidene fluoride membranes (Millipore). Next, membranes were blocked with 5% (M/V) skimmed milk for an hour and were incubated with primary antibodies including alpha-smooth muscle actin (α-SMA; Affinity, Changzhou, China), collagen I (Affinity), suppressor of cytokine signaling 1 (SOCS1; ABclonal Biotechnology, Shanghai, China), p38 (ABclonal Biotechnology), and p-p38 (ABclonal Biotechnology) at 4°C overnight. Glyceraldehyde-3-phosphate dehydrogenase (Proteintech. Wuhan, China) was used as a loading control. All primary antibodies were diluted 1:1000 except for Glyceraldehyde-3-phosphate dehydrogenase (diluted 1:10,000). Subsequently, protein blots were incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:3000; Solarbio) at 37°C for an hour. The relative protein levels were visualized by employing horseradish peroxidase chromogenic substrate (Solarbio). The images were captured by WD-9413B gel imaging system (Liuyi Biotechnology, Beijing, China).

Cell lysates were separated using 10% SDS-PAGE and transferred onto Immobilon PVF-membranes (Millipore, USA) by electrotransfer. A protein size ladder (Thermo Fisher Scientific, USA) was used for size comparison. The primary antibodies used were as follows: cleaved caspase-3 (Cell Signaling Technology, USA), GSDME (Abcam, UK), caspase-1, GSDMD, IL-1β, IL-18 (all Abcam) and glyceraldehyde-3-phosphate dehydrogenase (ProteinTech Group, USA). Secondary horseradish peroxidase-coupled antibodies (KPL, USA) and Super ECL Plus chemiluminescent detection substrate (US Everbright and Immobilon Western) were used for detection. The protein bands were analyzed using an ECL chemiluminescent detection system (Bio-Rad, CA) and band intensity was quantified using ImageJ software version 1.8.0 (https://imagej.nih.gov/ij/).