Anti phospho p65 ser536

The VK2/E6E7 vaginal epithelial cells were seeded onto six-well tissue culture plates and incubated in supplement-free KSFM for 12 h and then infected with Candida cells with a multiplicity of infection (MOI) of 5. EGFR inhibitors were added to the host cells 2h before the fungal stimulation. At various time points, the epithelial cells were rinsed with cold PBS and lysed using a modified RIPA lysis buffer containing protease (Cell Signaling Technology) and phosphatase (Sigma-Aldrich) inhibitors, left on ice for 30 min. The cells were collected by centrifugation and supernatants were assayed for total protein. 20 ug of the protein was separated by SDS-PAGE and the proteins were detected by immunoblotting with specific antibodies, including anti-phospho-EGFR Tyr1068 (#3777), anti-phospho-c-Fos Ser32 (Cell signaling; #5348), anti-phospho-p65 Ser536 (Cell signaling; #3033), anti-phospho-JNK Thr183/Tyr185 (Cell signaling; #9255),anti-phospho-Erk1/2 Thr202/Thr204 (Cell signaling; #4370), anti-phospho-p38 Thr180/Tyr182 (Cell signaling; #4511). For the extraction of total protein from vaginal tissue, half of the dissected vagina was firstly grinded and then lysed using Minute™ Total Protein Extraction Kit for Animal Cultured Cells/Tissues (SD-001/SN-002, invent biotechnologies, America) at 4°C. After quantitation, the tissue protein was separated by SDS-PAGE and detected as above.

For western blotting, polypeptides were resolved by SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad). Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies. Chemoluminescence reagent (Bio-Rad) was used as a substrate and the signal was scanned using the Chemidoc and accompanying software (Bio-Rad) to quantify the intensity of the bands as a measure of the amount of protein of interest in the blot. The relative amount was determined by calculating the ratio of each protein over that of the density measured for the housekeeping protein β-actin. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 6.

Dulbecco’s modified Eagle’s medium (DMEM)/F-12 and fetal bovine serum were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). BioTrace^TM NT membrane was purchased from Pall Life Sciences (Ann Arbor, MI, USA). The enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Perkin Elmer (Waltham, MA, USA). Anti-phospho-EGFR (Tyr¹¹⁷³, Cat#4407), anti-phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸², Cat#9211), anti-phospho-JNK1/2 (Thr¹⁸³/Tyr¹⁸⁵, Cat#9255), anti-phospho-FoxO1 (Ser²⁵⁶, Cat#9461), and anti-phospho-p65 (Ser⁵³⁶, Cat#3033) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH (Cat#MCA-1D4) was purchased from EnCor Biotechnology (Gainesville, FL, USA). AG1478, SP600125, and Tanshinone IIA were purchased from Enzo Life Science (Farmingdale, NY, USA). AS1842856 was obtained from EMD Millipore (Billerica, MA, USA). Helenalin was purchased from Cayman Chemical (Ann Arbor, MI, USA). Recombinant human TNF-α protein was purchased from R&D Systems (Minneapolis, MN, USA). TRIzol reagent, enzymes, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The following reagents were used: Doxorubicin (Sigma), Etoposide (Sigma), Z-IETD-FMK (Santa Cruz), LCL161 (Selleckchem), anti-PAR-4 (sc-1807, Santa Cruz), anti-PAR-4 (ab5787, Abcam), anti-GAPDH (sc-32233, Santa Cruz), anti-caspase-8 (#9746, Cell Signaling), anti-cleaved caspase-8 (#9496, Cell Signaling), anti-PARP-1 (#9542, Cell Signaling), anti-cIAP1 (#7065, Cell Signaling), anti-cIAP2 (#3130, Cell Signaling), anti-XIAP (#2045, Cell Signaling), anti-phospho-p65 Ser536 (#3033, Cell Signaling), anti-IκBα (#9242, Cell Signaling), anti-GFP (Rockland), anti-rabbit-HRP (P0448, DAKO), anti-mouse-HRP (P0447, DAKO), anti-rabbit-Alexa-Fluor-555 (A-31572, Thermo Fisher) and anti-rabbit-Alexa-Fluor-488 (A-21206, Thermo Fisher).