Anti phospho egfr tyr1068

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.

PD98059 and Ly294002 were purchased from Cell Signaling Technology (Danvers, MA). Recombinant human HB-EGF was purchased from R&D Systems (Minneapolis, MN). The following primary antibodies were used: anti-GPC1 antibody from Atlas antibodies (Stockholm, Sweden); anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-p70S6K (Thr389), anti-p70S6K, anti-phospho-p44/42 (Thr202/Tyr204), anti-p44/42, anti-Bak, anti-Bim, anti-Bcl-w and anti-phospho-EGFR (Tyr1068) from Cell Signaling Technology; anti-GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-EGFR from BD Transduction Laboratories (San Jose, CA).

Immunoblot analysis was performed as previously described [18 (link)]. Total cell lysates were prepared and subjected to immunoblot analysis using the following antibodies: anti-ERK1/2 (1:1000), anti-phospho-ERK1/2 (1:1000), anti-MAPK/ERK kinase 1/2 (MEK1/2) (1:1000), anti-phospho-MEK1/2 (1:1000), anti-phospho-Raf-1 (Ser-338) (1:1000), anti-EGFR (1:1000), anti-phospho-EGFR (Tyr-1068) (1:1000) (Cell Signaling Technology Inc., Beverly, MA, USA), anti-Raf-1 (1:1000) (BD Transduction Laboratories, BD Biosciences Inc., San Jose, CA, USA), or anti-involucrin (1:1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The luminescent signals were analyzed using an LAS-4000 image analyzer (Fuji Film Co., Tokyo, Japan). Immunoblot band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA).

Cells were fixed in 4% PBS‐paraformaldehyde for 15 min, incubated in 0.1% Triton X‐100 for 5 min on ice, then incubated in 0.2% Fish Skin Gelatine in PBS for 1 h, and stained for 1 h with an anti‐Phospho‐EGFR^Tyr1068 (1 : 100; Cell Signaling Technology, 3777). Protein expression was detected using Alexa Fluor (1 : 400; Molecular Probes, Eugene, OR, USA) for 20 min. TO‐PRO‐3 (Invitrogen, Waltham, MA, USA) was used to stain nucleic acids (1 : 2000).

Cells were cultured in serum-free medium for 24 h and then treated with gefitinib (40 nM) or/and estradiol (20 nM) for another 8 h. Total protein was extracted from cells using cell lysis buffer (Beyotime, China) supplemented with a protease inhibitor cocktail (Roche, Germany); protein concentration was determined using the BCA protein assay (Beyotime, Beijing, China). The following primary antibodies (1:1,000) were used: anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-AKT, anti-phospho-AKT (Ser473), anti-RPS6, anti-phosphor-RPS6 (Ser235/236), anti-P21, anti-CyclinD3, anti-cleaved-PARP (cPARP), and anti-beta-actin (Cell Signaling Technology, USA). Membranes were then incubated with peroxidase-linked anti-mouse or anti-rabbit secondary antibodies (1:5,000; Cell Signaling Technology, USA) for 2 h at room temperature. The expression levels of indicated proteins were quantified using quantity one.

Western blot analysis was performed as described previously [21 (link)]. The primary antibodies used for Western blot analyses included anti-phospho-Src (Tyr418) (Invitrogen, Carlsbad, CA, USA), anti-phospho-FAK (Tyr576) (Invitrogen), anti-FAK (Invitrogen), anti-GAPDH (Invitrogen), anti-phospho-EGFR (Tyr1068) (Cell Signaling, Beverly, MA, USA), anti-phospho-STAT3 (Tyr705) (Cell Signaling), anti-phospho-PI3K (Tyr458) (Cell Signaling), anti-AKT (Cell Signaling), anti-phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling), anti-SAPK/JNK (Cell Signaling), anti-phospho-Paxillin (Tyr118) (Cell Signaling), anti phosphor-p130Cas (Tyr410) (Cell Signaling), anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT3 (Santa Cruz Biotechnology), anti-PI3K (Santa Cruz Biotechnology), anti-phospho-MEK1/2 (Ser218/Ser222) (Santa Cruz Biotechnology), anti-MEK (Santa Cruz Biotechnology), anti-phospho-ERK (Tyr204) (Santa Cruz Biotechnology), anti-ERK2 (Santa Cruz Biotechnology), anti-Paxillin (Santa Cruz Biotechnology), anti-p130 Cas (Santa Cruz Biotechnology), anti-phospho-AKT (Ser473) (Millipore, Billerica, MA, USA)

Sodium dodesyl sulfate (SDS) polyacrylamide gels (Bio-Rad, Hercules, CA) were loaded with 40 μg total protein per lane; following electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad), which were incubated with Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, followed by overnight incubation at 4°C with anti-ALK (C26G7), anti-phospho-ALK (Tyr1604), anti-phospho-EGFR (Tyr1068), anti-STAT3(79D7), anti-phospho-STAT3 (Y705), anti-AKT, anti-phospho-AKT (Ser473), anti-ErbB4 (111B2), anti-phospho-ErbB4 (Tyr1284), anti-MET (25H2), anti-phospho-MET (Y1234/Y1235) (3D7), or anti-β-actin (13E5) antibodies (1:1,000 dilution each; Cell Signaling Technology, Danvers, MA), or with anti-human EGFR (1 μg/mL), anti-human/mouse/rat extracellular signal-regulated kinase (Erk)1/Erk2 (0.2 μg/mL), or anti-phospho-Erk1/Erk2 (T202/Y204) (0.1 μg/mL) antibodies (R&D Systems). After washing 3 times, the membranes were incubated for 1 h at room temperature with secondary antibodies (horseradish peroxidase-conjugated species-specific antibodies).
Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate Enhanced Chemiluminescent Substrate (Pierce, Osaka, Japan). Each experiment was independently carried out at least 3 times.