Mouse anti human gapdh

Antibodies were as follows: anti-transferrin receptor CD71 conjugated with phycoerythrin (PE) (clone YDJ1.2.2; Beckman Coulter), mouse control isotype IgG1 (BD Biosciences), mouse anti-human clathrin heavy chain (Abcam), rabbit anti-human dynamin-2 (Interchim), rabbit anti-human ATP6V1β2 (Abcam), mouse anti-human caveolin-1 (Santa Cruz), rabbit anti-human Pak1 (Santa Cruz), rabbit anti-human Rac1 (Santa Cruz), mouse anti-human cortactin (Santa Cruz), antitransferrin conjugated with Alexa Fluor 555 (Life Technologies), mouse anti-human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Abcam), fluorescein isothiocyanate (FITC), or Texas Red-conjugated goat anti-mouse IgG (Dako, CA, United States), and anti-mouse or anti-rabbit horseradish peroxidase (HRP)-coupled secondary antibodies (Agilent Technologies). The anti-IL-2 receptor β-chain monoclonal antibody 561 was previously described (64 (link)). Mouse anti-E protein 2D12 were produced from the hydridoma cell line ATCC CRL-1689. Mouse anti-E 4G2 and ascites from mice inoculated with the French neurotropic (FNV) strain of YFV were produced by RD Biotech, France. E16 anti-WNV E was previously described (65 (link)). Dynasore (Sigma-Aldrich D7693) was used at the indicated concentration by treating the cells 30 min prior to infection. In these experiments, dimethyl sulfoxide (DMSO) was used as a control treatment.

To assess EGFR expression, total protein samples were extracted from HCC827 and NCI-H520 cells in lysis buffer (RIPA) containing protease inhibitor (PMSF). The resulting lysates were centrifuged at 13.2×10³ rpm for 15 min and the supernatants were collected. The protein concentrations were measured using the BCA protein assay kit (Solebo biotech Ltd). SDS–PAGE and Western blotting were performed using 80 μg of proteins. The lysates were resolved by electrophoresis (70 V for 25 min and 110 V for 1.5 h) and transferred onto NC membranes. After blocking in 5% non-fat milk for 2 h, the blots were incubated with the first antibody: rabbit anti-human EGFR monoclonal (1:1000, Abcam) and mouse anti-human GAPDH (1:1500, Abcam) were used as a loading control overnight at 4 °C. The blots were washed and incubated with the second antibody: goat anti-rabbit IgG HRP-linked antibody (1:5000, Cell Signaling Technology); goat anti-mouse antibody (1:5000, Cell Signaling Technology) at room temperature for 1.5 h. Western blot bands were captured by the ECL Western blotting detection system (BD). GAPDH was used as a loading control. After development, the films were scanned with BIO-RAD Gel Doc XRS⁺. The images were opened and analyzed by ImageLab (BIO-RAD) software. Three samples of each tumor cell type were prepared for Western blot to obtain semi-quantitative data for statistical analyses.

Panc-1 and MiaPaCa-2 cells were collected and lysed for 15 min on ice in RIPA buffer (Beyotime Institute of Biotechnology). Quantification of protein concentration was measured by bicinchoninic acid kit (Beyotime Institute of Biotechnology). A total of 30-50 µg protein was loaded per lane, separated by SDS-PAGE on 8-12% gels and transferred to 0.45-µm polyvinylidene difluoride (PVDF) membranes (Immobilon-P; MilliporeSigma). Subsequently, 5% skim milk (cat. no. 70166; Sigma-Aldrich; Merck KGaA) was used to block PVDF membranes for 1 h at room temperature. The membranes were incubated with primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies were then used to incubate membranes at room temperature for 1 h. Protein expression was detected using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.). The following primary antibodies were used: Rabbit anti-human HNF1A (1:1,000; cat. no. ab96777; Abcam), 53BP1 (1:1,000; cat. no. ab87097; Abcam), mouse anti-human GAPDH (1:2,000; cat. no. abs830030; Absin Bioscience, Inc.) The secondary antibodies were goat anti-rabbit IgG-HRP (1:10,000; abs20002) and goat anti-mouse IgG-HRP (1:10,000; cat. no. abs20001) (both from Absin Bioscience, Inc.).