Mouse anti egfr

Cells were lysed in RIPA buffer (Cell Signaling; Danvers, MA, USA) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Total protein extracts were run on SDS-PAGE and blotted onto nitrocellulose. Blots were probed overnight. The following antibodies were purchased from Cell Signaling: Rabbit anti-phospho-IGF-1 receptor β (Tyr1131) (#3021, 1:200); rabbit anti-IGF-1 receptor β (#3018, 1:250); rabbit anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9101, 1:1000); rabbit anti-p44/42 MAPK (Erk1/2) (#9102, 1:1000); rabbit anti-phospho-Akt XP (Ser473) (#9018, 1:1000); rabbit anti-AKT (#9272, 1:1000); rabbit anti-phospho-EGFR (Tyr1148) (#4404, 1:200); and mouse anti-EGFR (#2239, 1:200). Mouse anti-β-actin was purchased from Sigma-Aldrich (AC-15, 1:15,000). All primary antibodies were diluted in 5% BSA/TBS-T. Goat anti-mouse secondary IRDye 800 antibody (#926-32210, 1:10,000) was purchased from Li-Cor Biosciences (Lincoln, NE, USA). Goat anti-rabbit alexa-fluor 680 secondary antibody (#1027681, 1:10,000) was purchased from Invitrogen (Grand Island, NY, USA). Protein bands were detected using the Odyssey Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).

Following treatment with AZD8931 at the indicated concentration and time points, immunoblotting was performed as previously described
[15 ]. In brief, cells were lysed in 1× lysis buffer (Cell signaling), and then the supernatant was collected by centrifuging at 10,000 rpm for 10 min at 4°C. Protein concentration was determined using the BCA protein assay reagent kit (Pierce, Rockford, IL). Equal amounts of protein from cell lysates were resolved by SDS-PAGE electrophoresis. The membranes were incubated at 4°C overnight with the following antibodies: mouse anti-EGFR (1:1000; Cell Signaling), rabbit anti-AKT and rabbit anti-phospho-AKT (1:1000; Cell Signaling), mouse anti-β-actin (1:5,000; Santa Cruz). After incubation with anti-mouse IgG horseradish peroxidase conjugated secondary antibody (1:5,000; Amersham Pharmacia Biotech), immunoreactive proteins were visualized by the enhanced chemiluminescence reagents.