P egfr tyr1068

Cells were lysed with Nonidet P-40 (1%, Sigma) that included protease inhibitor cocktail (Roche) and then centrifuged for 15 min at 14,000 r.p.m. at 4°C. Cell protein lysates (50-100 μg) were dissolved in loading buffer (62.5 mM Tris-HCl pH 6.8, 10% Glycerol, 2% SDS, 0.01% Bromophenol Blue, 100mM DTT), and separated by SDS-polyacrylamide gel electrophoresis. Antibodies used for western blotting were: Tf Receptor (Abcam ab38171, 1:250); EGFR (Millipore 06-847, 1: 250); p-EGFR, Tyr1068 (Cell Signaling 2234, 1:1000); ERK1/2 (Sigma M 5670, 1:2000); pERK1/2, Thr183 (Sigma M 7802, 1:500); Cyclin D1 (Sigma C 7464, 1:200); AKT (Cell Signaling 9272, 1:1000); and pAKT, Ser 473 (Cell Signaling 4056, 1:1000). Loading controls used were Tubulin (Sigma T 9026, 1:1000) and GAPDH (Sigma G 9295, 1:20,000).
Cells used for degradation assays were plated at 2.5 × 10⁵ cells/well in a 6-well tissue culture dish and allowed to adhere overnight. The following day cells were growth factor starved for 2 hrs in the presence of 50 μM cycloheximide (Sigma). These conditions were maintained for the duration of the experiment. Following, 20 ng mL^-1 EGF (PeproTech) was added to the media for the indicated amounts of time. Cells were then chilled on ice, washed with PBS, and protein lysates were collected for immunoblotting. Blots were cropped to show relevant bands.

Antibodies against EGFR, p-EGFR (Tyr1068), AKT, p-AKT (Thr308), cleaved PARP (C-PARP) and GAPDH were purchased from Cell Signaling Technology, and those against CXCR7, and VEGFR2 from Abcam, Inc. Densitometry analysis for WB was performed using FIJI imaging software.^{25 (link)} Expression levels of p-EGFR and p-AKT (308) were normalized to total EGFR and AKT levels, and VEGFR2 to GAPDH.