Minute total protein extraction kit for animal cultured cells tissues

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.

HEK 293T or MDCK cells were grown to 80% confluence and transfected with different gene expression plasmids or empty vector. Cells were infected with CIV (or left uninfected) 24 h after transfection. After 24 h, the cells were lysed using Minute Total Protein Extraction Kit for Animal Cultured Cells/Tissues (Invent Biotechnologies, Plymouth, MN, USA). Lysates were collected and centrifuged for 30 s. A total of 30 μg of each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane that was blocked for 15 min with QuickBlock Blocking Buffer for Western Blot (Beyotime) and then incubated overnight at 4 °C with primary antibodies, followed by a 1-h incubation with secondary antibody. Protein bands were visualized by Odyssey Sa (Li-cor, Lincoln, NE, USA).

Coimmunoprecipitation (CoIP) was performed using the Minute™ Total Protein Extraction Kit for Animal Cultured Cells/Tissues (Invent Biotechnologies, United States). Cells for CoIP were treated with DMSO or PEITC for 24h. SN-002 buffer was used to lyse the cells, and a BCA protein assay kit (Solarbio, China) was used to measure protein concentrations. We incubated lysates with antibodies overnight at 4°C before incubating them with protein A agarose for 2 hours. We washed beads three times with 1× PBS and boiled them with SDS-PAGE Sample Loading Buffer (5×, with DTT) (Leagene Biotecnology, China). For the analysis of protein interactions between immunoprecipitated samples, immunoblotting was performed. The anti-p53 (Proteintech, #10442-1-AP), anti-PAb240 (Merck Millipore, #OP29L), anti-PAb1620 (Merck Millipore, #OP33) and anti-p73 (abcam, #ab215038) were used.