Phospho p38 antibody

ER-negative breast cancer cell lines MDA-MB-231, SKBR3, BT-474, and T47D were obtained from the American Type Culture Collection (Manassas, VA, USA). Recombinant adenoviruses, ad-mgp96 expressing mgp96, and control adenoviruses ad-pDC312 were created by our lab. The ER-α36-knockdown cell line, MDA-MB-231-ER-α36i, and MDA-MB-231-mock cell line, the ER-α36-negative breast cancer cell line MCF7-10A, ER-α36 antibody, E2β and BSA-E2β were generous gifts from Beijing Shenogen Biomedical Co. Ltd. Gp96 polyclonal antibody and Protein G were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). The gp96 monoclonal antibody (mAb) was generated in our lab. ERK antibody, Phospho-ERK antibody, p38 antibody, and Phospho-p38 antibody were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). The remaining antibodies were obtained from Zhongshan Goldenbridge Biotechnology (Beijing, China). Cycloheximide (CHX) and MG132 were from Beyotime Institute of Biotechnology (Shanghai, China). Glutathione Sepharose 4B was from GE Healthcare Life Sciences (Little Chalfont, Buckinghamshire, United Kingdom). The protein cross-linkers DTSSP and BS3 were purchased from Thermo Scientific (Waltham, Massachusetts, USA).

DMEM, fetal bovine serum (FBS) (Gibco/invitrogen, Carlsbad, CA, USA), a Cell Counting Kit-8 assay (Dojindo Lab, Kumamoto, Japan), β-glucan kit (Megazyme International, Wicklow, Ireland), Phagocytosis Assay Kit (Cayman, MI, USA), cyclophosphamide (Sigma Aldrich, St.Louis, MO, USA) β-1,3-glucan (Sigma Aldrich, St.Louis, MO, USA) and cordycepin (Sigma Aldrich, St.Louis, MO, USA) were purchased. Phospho-Lyn antibody, Lyn antibody, phospho-Syk antibody, Syk antibody, phospho-ERK antibody, ERK antibody, phospho-p38 antibody, p38 antibody, phospho-JNK antibody, JNK antibody, NFκB antibody, phospho-IκB antibody and IκB antibody were obtained from Cell signaling Technology Inc. (Danvers, MA, USA). β-Actin antibody was obtained from Santa Cruz (Dallas, TX, USA).

Total RNA was extracted from whole tracheas using the Direct-zol RNA MiniPrep Kit (Zymo Research). cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad), and quantitative RT-PCR was performed with iQ SYBR Green Supermix (Bio-Rad) using a StepOne Plus system (Applied Biosystems). For primer sequences see Table S2.
Western blot analysis was performed on protein extracts from total trachea, epithelium and mesenchyme. Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked for 1 h with 5% (w/v) dried milk in PBS containing 0.1% Tween 20, and incubated with phospho-Smad1/5/8 antibody (1:1000; Cell Signaling, 9511), phospho-p38 antibody (1:2000; Cell Signaling, 4511), phospho-SAPK/JNK (Thr183/Tyr185) (G9) antibody (1:1000; Cell Signaling, 9255) and β-actin antibody (1:3000; Abcam, ab8226) in blocking buffer overnight at 4°C, followed by HRP-conjugated secondary antibody (Bio-Rad). Proteins were visualized using the ECL detection system (FEMTOMAX-110, Rockland Immunochemicals). The phospho-Smad1/5/8 band was validated by a positive control (HMEC1 cell treated with Bmp9) and phospho-p38 and phospho-Jun bands by molecular weight.

MACS-purified CD4⁺CD25⁺ T cells were stimulated with TNF (100 ng/mL), with or without selected inhibitors [SB203580 (SB), Bay 11-7082 (Bay), Sulfasalazine (Sul)] for 30 min. The cells were homogenized in RIPA buffer containing a cocktail of proteinase and phosphatase inhibitors. Protein samples were separated on a SDS-PAGE gradient gel (4–12% Bis-Tris protein gel; Thermo Fisher Scientific) and transferred to PVDF membranes. The blots were blocked with 5% BSA for 1 h and incubated with phospho-p38 antibody (1:1,000; Cell Signaling Technology) and phospho-NF-κB p65 antibody (1:1,000; Cell Signaling Technology) overnight at 4°C. The blots were then incubated in HRP-conjugated secondary antibody (1:3,000) for 1 h at room temperature, developed in ECL solution (Thermo Fisher Scientific) for 1 min, and exposed by G-Box imager. The blots were then incubated in stripping buffer (Thermo Fisher Scientific) at 37°C for 15 min and reprobing with IκBα antibody (1:1,000; Cell Signaling Technology) or p38 antibody (1:1,000; Cell Signaling Technology) or NF-κB p65 antibody (1:1,000; Cell Signaling Technology) or GAPDH antibody (1:3,000; Cell Signaling Technology).