Phospho erbb 1

Cells were washed with PBS and harvested using RIPA Buffer (R0278, Sigma UK) with 1× proteinase inhibitors. Protein quantification was performed using the Bradford reagent (Sigma-Aldrich), according to the manufacturer’s instructions. Cell extracts (25 μg) were run on a NuPAGE® Novex® 4-12% Bis-Tris Gel, 1.5 mm and transferred to a nylon membrane. The membrane was blocked overnight (4°C in PBS with 0.1% Tween and 5% milk powder) and probed using the following antibodies: Phospho-Stat3 (Tyr705) (1:1000 #9145, Cell Signaling), Stat3 (1:1000 #4904, Cell Signaling), p-ERK (1:1000 sc-7383, Santa Cruz), ERK (1:1000 #9102, Cell Signaling), phospho-ErbB-1 (1:1000 #2220, Cell Signaling), ErbB-1 (1:1000 2232, Cell Signaling), phospho-ERbB-4 (1:500 #3790, Cell Signaling), ERbB-4 (1:1000 #4795, Cell Signaling), TNFα (1:1000 ab6671, Abcam), Jagged1 (1:1000 SC-8303, Santa Cruz), α-tubulin (1:2000 sc-8035, Santa Cruz), Lamin A/C (1:2000 #2032, Cell Signaling), β-Actin (1:5000 A5316, Sigma). A rabbit or mouse horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Chalfont St Giles, UK) incubation allowed visualization using enhanced chemiluminescence (ECL) (GE Healthcare). Protein concentration equivalence was confirmed by anti–β-Actin antibody.