Mouse anti β actin monoclonal antibody

RNAi knockdown of Dab2 and Numb in RPTPalpha MEF was performed using ON-TARGETplus Mouse Dab2 and Numb siRNA purchased from Dharmacon (Lafayette, CO, USA). siRNA pools were transfected by electroporation, either separately or in combination, at a final concentration of 50 nM and a cell density of 10⁷ cells ml⁻¹ using the manufacturer's protocol. Efficiency of RNAi knockdown was analysed after 36 h of transfection by western blot analysis using antibodies specific to Dab2 and Numb. Rabbit monoclonal antibodies for Dab2 and Numb (#12906 and #2756, respectively) were purchased from Cell Signaling Technology (Danvers, MA, USA) and were used at a dilution of 1:1,000 for immunodetection. β-actin was used as an internal loading control and was detected using mouse monoclonal anti-β-actin antibody purchased from Abcam (Cambridge, MA, USA). The knockdown efficiency was found to be 70–90%. The full blots are available in Supplementary Fig. 16.

Protein extraction and western blot analysis were carried out as described (Miao et al. 2017 (link)). Briefly, lysate was obtained from specimens of pancreatic tumors and peripancreatic tumor tissues. Specimens were ground using a low-speed tissue homogenizer. All operations were performed on ice. Homogenates were centrifuged (12,000 rpm, 15 min, 4 °C), and the supernatant was collected. The protein content was measured with a BCA protein assay. Subsequently, protein was denatured at 99 °C for 5 min. Equal amounts of protein (30 μg/sample) were loaded onto sodium dodecyl sulfate-polyacrylamide gels, electrophoresed, and transferred onto polyvinylidene difluoride membranes. Membranes were incubated overnight at 4 °C with mouse monoclonal anti-β-actin antibody (1:3000, Abcam, ab5694) and rabbit monoclonal anti-mast cell tryptase antibody (1:500, Abcam, ab151757), followed by anti-mouse IgG-horseradish peroxidase (HRP) (1:5000, Cell Signaling Technology, #7076) and anti-rabbit IgG-HRP (1:5000, Cell Signaling Technology, #7074) antibodies, respectively, for 2 h at room temperature. A chemiluminescence reagent kit (ECL, Bio-Rad, Hercules, CA, USA) was used to detect the immunoreactive bands. Protein bands were normalized to those of β-actin. Image-Pro Plus 6.0 software was used to quantitate the protein content.

All reagents used in this study were purchased from Sigma-Aldrich Co. (St. Louis, MO) unless otherwise noted. The rabbit polyclonal anti-ADD1 antibody (Catalog# NBP1-48,611) and the mouse monoclonal anti-TPX2 antibody (Catalog# NBP2-67,265) were purchased from Novus Biologicals. The rabbit polyclonal anti-p-ADD1 (S726) antibody (catalog# orb14892) was purchased from Biorbyt. Anti-α-tubulin-FITC (Catalog# F2168) and anti-γ-tubulin (Catalog# T6557) mouse monoclonal antibodies were obtained from Sigma-Aldrich. The mouse monoclonal anti-β-actin antibody (Catalog# ab49900) was purchased from Abcam. Alexa Fluor® 594-conjugated goat anti-rabbit IgG (H + L) (Catalog# A-11037) and Alexa Fluor® 647-conjugated goat anti-rabbit IgG (H + L) (Catalog# A-31633) were produced by Thermo Fisher Scientific Co.; TRITC conjugated goat anti-mouse IgG (H + L) (Catalog# ZF-0313) was purchased from Zhongshan Golden Bridge Biotechnology Co., LTD. Goat anti-rabbit IgG (Catalog# 1,706,515) and goat anti-mouse IgG (Catalog# 1,706,516) horseradish peroxidase (HRP)-conjugated antibodies were purchased from Bio-Rad.

The following materials and antibodies were purchased: LPS from Escherichia coli 0128:B12 (Sigma-Aldrich); LPS from P. gingivalis (InvivoGen); recombinant human HSP70 (Stressgen; endotoxin activity, <0.05 endotoxin units (EU)/μg); recombinant bovine HSC70 (Stressgen; <0.05 EU/μg); recombinant bovine HSC70 ATPase fragment (Enzo Life Sciences; 0.1 EU/μg); V8 protease (Roche); 15-deoxyspergualin (DSG) (Spanidin® Inj.; Nippon Kayaku); mouse monoclonal anti-TLR4 (HTA125) and anti-TLR2 (TL2.1) antibodies (Hycult Biotechnology); mouse monoclonal anti-CD14 (MY4) antibody (Beckman Coulter); rabbit polyclonal anti-p44/42, anti-p38, anti-phosphorylated p38, anti-phosphorylated IκB-α, mouse monoclonal anti-phosphorylated p44/42 antibodies (Cell Signaling); mouse monoclonal anti-JUN-N-terminal protein kinase (JNK) (D-2), anti-phosphorylated JNK (G-7), anti-IκB-α (H-4) antibodies (Santa Cruz Biotechnology); and mouse monoclonal anti-β-actin antibody (Abcam). Endotoxin levels of all materials used were determined using a ToxinSensor chromogenic LAL endotoxin assay kit (GenScript), and the resultant level was low (~0.1 EU/μg). The LPS, bovine serum albumin (BSA), HSP70, HSC70, and the HSC70 ATPase fragment were heated at 95°C for 20 min.