Pi3k 20584 1 ap

Recombinant rat IL-1β (501-RL-010) was obtained from R&D systems (Minneapolis, MN, USA). Antagonist AZ3451 was obtained from MedChem Express (Monmouth Junction, NJ, USA). The p38 MAPK inhibitor SB203580, the NF-κB nuclear translocation inhibitor JSH-23 and the P13K/AKT inhibitor LY294002 were obtained from Selleck chemicals (Houston, TX, USA). The antibodies against P38(#8690), phospho-P38(#4511), ERK ½ (#4695), phospho-ERK ½ (#4370), JNK (#9258), phospho-JNK (#9255), phospho-P65(#3033), phospho-IKBα (#2859), phospho–IKKαβ (#2697), IKKβ (#8943), AKT (#4685), Phospho-AKT (#4060), Phospho–PI3K (#17366), mTOR (#2983), Phospho-mTOR (#5536), COX2(#12282), Atg5 (#12994), Atg7(#8558), Atg12(#4180), Beclin1(#3495), LC3A/B (#12741), Cleaved Caspase-3 (#9664) and p16INK4A (#80772) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against Collagen Type II(15943-1-AP), MMP1 (10371-2-AP), IKBα (10268-1-AP), P65 (10745-1-AP), Histone-H3(17168-1-AP), Cytochrome C (10993-1-AP), Caspase 3 (19677-1-AP), Bax (50599-2-Ig), Bcl-2 (12789-1-AP) and PI3K (20584-1-AP) were purchased from Proteintech Group (Wuhan, China). Antibodies against iNOS (ab3523), MMP13 (ab39012), PAR2 (ab180953), SOX9 (ab185966) and Aggrecan (ab36861) were supplied by Abcam (Cambridge, UK). Antibodies against ADAMTS5 (BA3020) and GAPDH (BM3876) were obtained from Boster (Wuhan, China).

SAF was isolated from a fungal strain in a lab from the Department of Pharmaceutical Sciences of Binzhou Medical University. SAF was dissolved in dimethyl sulfoxide (DMSO) and was stored at −20 °C as a stock solution. MARCH1 (bs-9335, Bioss, Beijing, China), phospho-AKT Ser473 (Sikh Association of Baltimore, Randallstown, MD, USA), total AKT (21054, SAB), GAPDH (10494-1-AP, Proteintech, Wuhan, China), PI3K (20584-1-AP, Proteintech, Tokyo, Japan), β-catenin (51067-2-AP, Proteintech), Mcl-1 (16225-1-AP, Proteintech), Bcl-2 (12789-1-AP, Proteintech), cleaved caspase-3 (9661, Commonwealth Soap & Toiletries, Fall River, MA, USA), cleaved caspase-7 (8438 CST), MG 132 (HY-13259, MedChem Express, Monmouth Junction, NJ, USA) and secondary antibodies (peroxidase-conjugated goat anti-rabbit IgG; ZB-2301, ZSGB-BIO, Beijing, China) were obtained commercially.

Proteins were isolated from cells using lysis buffer (containing RIPA and 1% PMSF). Protein quantification was performed using a BCA protein detection kit (23229; Thermo Fisher Scientific). Protein samples of the same mass were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, United States), which were blocked with 5% skim milk and then incubated with specific primary antibodies. Caspase-3 (14220T, 1:1000), PARP (9532T, 1:1000), β-actin (3700S, 1:1000), GAPDH (5174T, 1:1000), AKT (4685S, 1:1000), p-AKT (4060T, 1:2000), PI3K (4249T, 1:1000), ERK (4695T, 1:1000) and p-ERK (4370T, 1:1000) were purchased from Cell signaling Technology. The anti-rabbit (SA00001-2, 1:10000), PI3K (20584-1-AP, 1:1000) and anti-mouse (SA00001-1, 1:10000) secondary antibody were purchase from Proteintech. FluorChem M system was used to quantify the positive bands representing proteins involved in the orchestrated immune responses (ProteinSimple, San Jose, CA, United States).