Trans blot turbo transfer

Nuclear and cytoplasmic protein extracts of cells incubated with PT-G/PT-BSA for 4 h were isolated using a commercial Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) and protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Protein preparations were heat-denatured at 95 °C for 5 min, loaded into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer (BioRad, Hercules, CA, USA). After transfer, membranes were blocked in 5% non-fat milk in Tris-buffered saline with 0.05% Tween (TBST) and incubated overnight at 4 °C with the following primary antibodies: anti-HDAC1 (1:10,000 dilution; ABCAM, Cambridge, UK), anti-ELK1 (1:500), anti-NFκB p10550 (1:400), anti-CREB1 (1:1000) anti-IRF1 (1:1000) and anti-α-tubulin (1:5000 dilution, Sigma-Aldrich) followed by an incubation of 1 h with HRP (horseradish peroxidase) conjugated anti-rabbit Immunoglobulin G (IgG) (1:2000) and anti-mouse IgG (1:1000) secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Proteins were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) on a ChemiDoc MP system.

Whole cell protein extracts were obtained using RIPA buffer (Tris, pH 7.4, 50 mM NaCl, 150 mM, NP-40, 1%, Deoxycholic Acid, 0.5%, SDS, 0.1%) supplemented with protease (cocktail III; RPI, Mount Prospect, IL) and phosphatase inhibitors (Pierce, Hampton, NH). Lysates were homogenized through a 29 g needle and cleared by full speed centrifugation for 5 min. Protein quantification was performed using the BCA assay (Pierce, Hampton, NH). Protein extracts were mixed with Laemmli Sample Buffer (LSB) and separated using Mini-PROTEAN TGX (4–15%) gels (Bio-Rad, Hercules, CA) transferred using Trans-Blot Turbo Transfer (Bio-Rad, Hercules, CA), blotted according to standard protocols, detected by luminescence using ECL (GE Healthcare Hampton, NH) and imaged using a Bio-Rad ChemiDoc (Hercules, CA). For xenograft tumor protein extraction, tumors were crushed using a dry-ice, pre-chilled pestle and mortar, followed by 3 × 10 sec sonication in chilled conditions, before protein extraction was performed as above.

EGFR inhibition and intracellular signaling pathways were analyzed by western blot in all HNSCC cell lines and in the A431 (sensitive control) cell line. Cells were rinsed in ice-cold PBS then scraped and lysed in lysis buffer (50mM Tris pH7.6–8, 150mM NaCl, 5mM EDTA, 1mM Na3VO4, 10mM NaF, 10mM sodium pyrophosphate, 1% NP-40, and protease cocktail inhibitors). 20 μg of total protein were resolved by 10% SDS-PAGE and transferred to nitrocelulose membranes in TransBlot Turbo transfer (Bio-Rad). Primary antibody incubation was performed for human total EGF Receptor (D38B1), pEGFR-Tyr1068 (D7A5), HER2 (4290), pHER2- Tyr1221/1222 (2243), HER4 (4795), pHER4- Tyr1284 (4757), p44/42 MAPK (137F5); p.p44/42 MAPK-Thr202/Tyr204 (D13.14.4E); AKT(pan) (C67E7); pAKT-Ser473 (D9E); AKT1(C73H10) and β-tubulin (endogenous control), from Cell signaling (Danvers, USA). Both primary antibodies were diluted in TBS-T at 1:1000. After washing with TBS-T, membranes were incubated with Anti-rabbit secondary antibody Anti-rabbit (#7074, Cell Signaling Technology) at dilution 1:5000. Immune detection was done with ECL Western Blotting Detection Reagent (GE Healthcare), in automatic ImageQuant mini LAS4000 (GE Healthcare). Experiments were performed three times.