Beta actin mouse mab

Specific EGFR and phospho-EGFR (Tyr1068) rabbit monoclonal antibodies (mAb) were from Cell Signaling Technology, Inc. (Beverly, MA, United States). AG1478 were from Selleck Chemical., Co. (Houston, TX, United States). Beta-actin mouse mAb was from Proteintech (West Grove, PA, United States). Cy3- or FITC-conjugated goat anti-rabbit or anti-mouse IgG, peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgG were all from Proteintech. Recombinant hEGF was from Sigma Chemical., Co. (St. Louis, MO, United States). Prokaryotic recombinant protein NcMIC3 (rNcMIC3) and mouse antiserum specific for NcSAG1 was produced in our Laboratory. Full-length gene of N. caninum MIC3 was subcloned into the pET-28a (+) vector (TaKaRa, Japan) containing the His-tag, and the recombination pET-28a-NcMIC3 contains four EGF sequence repeats.

Differently cultured DCs were lysed in RIPA lysate with protease inhibitor for 30 min on ice. Then, the proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime Institute of Biotechnology, Beijing, China). After blocking with 5% nonfat milk at room temperature for 2 h, the membranes were hybridized with specific antibodies overnight at 4 °C. The membranes were then washed three times with TBST buffer and incubated with secondary antibodies at room temperature for 1 h. The results were visualized using a C-Digit chemiluminescence scanner (LI-COR, Lincoln, NE, USA). The antibodies used were as follows: NF-κB p65 (L8F6) mouse mAb (CST, Danvers, MA, USA), IκBα (L35A5) mouse mAb (CST, Danvers, MA, USA), beta Actin mouse mAb (Proteintech, Rosemont, IL, USA), HRP conjugated goat anti-mouse IgG (Proteintech, Rosemont, IL, USA), rabbit anti-porcine IκBα antibody (CST, Danvers, MA, USA), rabbit anti-porcine NF-κB p65 antibody (CST, Danvers, MA, USA), mouse anti-rabbit IgG-Alexa FluorR 647 (CST, Danvers, MA, USA), rabbit anti-porcine JAK2 antibody (Immunoway, Plano, TX, USA), rabbit anti-porcine p-JAK2 antibody (Immunoway, Plano, TX, USA), and rabbit anti-porcine STAT3 antibody (Immunoway, Plano, TX, USA).