Anti phospho c fos ser32

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.

The analysis of protein expression was performed as described previously [22 (link)] on cell lysates obtained with LDS sample buffer (Thermo Fisher Scientific). An amount of 20 µg of proteins were separated on Bolt™ 4–12% Bis-Tris mini protein gel (Thermo Fisher Scientific) and transferred to PVDF membranes (Immobilon-P membrane, Merck). Membranes were incubated for 1 h at RT in blocking buffer (Tris-buffered saline solution—TBS; 50 mM Tris-HCl pH 7.5, 150 mM NaCl) added with 3% non-fat dried milk and then overnight at 4 °C with primary antibodies in TBST (TBS + 0.5% Tween) containing 5% BSA. Anti-phospho-NF-κB p65 (Ser536), anti-phospho-IκBα (Ser32/36) anti-phospho-STAT1 (Tyr701), anti-phospho-STAT3 (Tyr705) and anti-phospho-c-Fos (Ser32) (1:2000, Cell Signaling Technology) rabbit polyclonal antibodies were employed. Anti-vinculin mouse monoclonal antibody (1:2000, Merck) was used as loading control. Immunoreactivity was visualized with SuperSignal™ West Pico PLUS Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Western Blot images were captured with iBright FL1500 Imaging System (Thermo Fisher Scientific) and analyzed with iBright Analysis Software (Thermo Fisher Scientific).