Akt1 c73h10

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.

Expression of Jab1/Csn5 (nuclear and cytoplasmic), p-Stat3, Akt (nuclear and cytoplasmic), CHOP, and Ki-67 was analyzed by immunohistochemical (IHC) techniques. An analysis was conducted of the paired tumor tissues used as well as the 4-μm continuous sections cut from the formalin-fixed, paraffin-embedded paired pNPC and rNPC specimens. IHC analyses of p-Stat3 (Tyr705) (M9C6, dilution 1:100; Cell Signaling Technology, Danvers, MA, USA), Jab1/Csn5 (Ab495, dilution 1:150; Abcam, Cambridge, UK), Akt1 (C73H10, dilution 1:200; Cell Signaling Technology), Ki-67 (M7240, dilution 1:80; Dako Cytoformation, Glostrup, Denmark), and CHOP (Ab27539, dilution 1:50; Abcam) were performed using the streptavidin-biotin-peroxidase complex technique. The procedure was as follows: conventional dewaxing hydration, 3% hydrogen peroxide treatment, then blocking by dropper solution for 20 min, antibody incubation at 4°C overnight, then goat anti-rabbit/goat anti-mouse IgG by dropper, incubation for 30 min at 37°C, followed by baths of diaminobenzidine and hematoxylin. Negative controls were analyzed along with each assay by replacing the primary antibody with phosphate-buffered saline.

Nbs were produced and extracted as described above. Anti-HA-agarose beads (A2095, Sigma Aldrich) were incubated with 10 μg periplasmatic extract for 1 h at 4°C with end-over-end rotation. The beads were washed with ice-cold Tris Lysis Buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100 pH 7.5), 1mM phenyl-metylsulfonyl fluoride (PMSF) and 200 μg/mL protease inhibitor cocktail) and incubated for 1 h at 4°C with end-over-end rotation with 1 mg crude lysate from MDA-MB-231 cells. A negative control where the EGFP Nb was added to the beads was included to determine non-specific binding of AKT to the agarose matrix and nanobody. The beads were washed three times with excess Tris Lysis buffer and heated to 95°C for 5 min in Laemmli Sample Buffer (5% SDS, 20% glycerol, 0,2% bromophenol blue, 5% β-mercaptoethanol, 65 mM Tris-HCl pH 6.8). Samples were analyzed by SDS-PAGE and Western blotting. The AKT isoforms were detected using isoform-specific Abs (AKT1 C73H10, AKT2 D6G4 and AKT3 62A8 from Cell Signaling Technology®) and the Nbs with an anti-HA-tag Ab (11583816001, Merck). ECL signal for the AKT isoforms was recorded using the Amersham Imager AI680 and the signal was quantified using Image Studio Lite (v5.2).