Phospho iκb

Directly conjugated monoclonal antibodies against CD8 and CD45RA and their isotypes and unconjugated CD8 antibodies were obtained from BD Biosciences (San Diego, CA). Anti-CCR7 and isotypes were purchased from R & D systems, Minneapolis, MN, and anti-CD3/CD28 was Life Technology, Camarillo, CA. TNF-α was obtained from Laguna Scientific, Laguna Niguel, CA. Antibodies to FLIP and IAP were purchased from Transduction Laboratories, San Diego, CA, and antibodies to phospho IKKα/β, phospho IκB, phospho JNK, phosphor TAK1 TAK1, and TAB2 were purchased from Cell Signaling Technologies, Inc. Beverly, MA. Antibodies to A20, TRAF2 and RIPK1 were obtained from Santa Cruz Biotechnology, Dallas, TX. In Situ Cell Death Detection Kit was purchased from Boehringer-Manheim, Indianapolis, IN.

Dulbecco’s modified Eagle’s medium (DMEM), penicilin-streptomycin, and trypsin-ethylenediaminetetra acetic acid (EDTA) were purchased from GIBCO-BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Gelatin, PI, and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific to caspase-3, caspase-8, caspase-9, cFILP, cIAP-2, Bcl-XL, COX-2, phospho-NF-κB, phospho-c-Jun, c-Jun, phospho-IKK, phospho-IκB, phospho-p38, p38, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, phospho-AKT, AKT, phospho-TAK, TAK, and RIP were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). NF-κB, PARP, MT1-MMP, uPAR, ICAM-1, and VEGF were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cyclin D1 was purchased from Milipore. The Can Get Signal^® Immunoreaction Enhancer Solution was purchased from Toyobo (Osaka, Japan). Matrigel was purchased from Becton Dickinson (Bedford, MA, USA). Dicentrine was ordered from Chengdu Biopurify Phytochemicals Ltd. (Sichuan, China).

Whole protein from cell lysates and lung tissues was extracted according to manufacturer's instructions (Keygene, China). Protein samples were separated on 10% SDS‐PAGE gels, and then, the proteins were transferred to PVDF membranes (Merck Millipore, Germany), blocked with 5% nonfat milk in TBST and incubated with primary antibody at room temperature for 4 hr or overnight. Then, membranes were incubated with HRP‐conjugated secondary antibody for one hour, and protein expression was detected using ECL (Merck Millipore, Germany). Primary antibodies against P16, P21, αSMA, and Collagen1α were purchased from Abcam (United Kingdom). Antibodies against PTEN, IKK, phospho‐IKK, IκB, phospho‐IκB, NF‐κB, and phospho‐NF‐κB were purchased from Cell Signaling Technology (USA).

Homogenized liver and spleen tissues, and splenocytes from spleen of WT mice and Jurkat T cells lysed by protein extraction solution (PRO-PREP, iNtRON Biotechnology) containing protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Total proteins (30 μg) were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk overnight and then incubated with primary antibodies (diluted 1 : 1000) for 1 h at room temperature. The membranes were immunoblotted with following primary antibodies: Phospho-STAT1, Phospho-JAK3, Phospho-JNK, Phospho-PLCγ, Phospho-IκB (Cell Signaling Technology, Beverly, MA, USA), STAT1, JAK3, JNK, PLCγ, IκB (Santa Cruz Biotechnology, Dallas, TX, USA). After washing with Tris-buffered saline containing 0.05% Tween-20 (TBST), the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1 : 3000) for 1 h at room temperature. Binding of antibodies to the PVDF membrane was detected with enhanced chemiluminescence solution (Amersham Bioscience, Buckinghamshire, UK) and X-ray film (AGFA, Mortsel, Belgium).

Protein extracts were obtained through standard protocols [27 (link)]. Western blot analyses were performed as described previously [27 (link)]. Specific primary antibodies against COL1A1, COL3A1, Fibronectin, α-SMA, ERK, IκB, phospho-IκB, JNK, phospho-JNK (Cell Signaling Technology, Danvers, MA, USA), TLR4, p-ERK, p65 and SDF-1α (Abcam) were used. β-actin (Abcam) and Lamin A (Santa Cruz) were used as loading controls for total protein and nuclear protein, respectively.

Actinomycin D, LPS, Rotenone, Antimycin, Oligomycin, Etomoxir and antibodies against β-actin were purchased from Sigma-Aldrich, USA, and Chloramphenicol were obtained from Wako Pure Chemicals, Japan. Actinonin was purchased from Enzo Life Sciences, Germany. Polyclonal antibodies against mouse p32 were raised in our laboratory. Antibodies against p38, phospho-p38, Erk1/2, phospho-Erk1/2, NFκB p65, phospho-NFκB p65, IκB, phospho-IκB, ATF4 were purchased from Cell Signaling Technology, USA. Anti-COX1 antibodies and anti-B23 antibodies were from Thermo Fisher scientific, USA. Total OHPHOS rodent antibodies cocktail were from Abcam, USA.