Journal: Clinical cancer research : an official journal of the American Association for Cancer Research
Article Title: Low expression of the E3 Ubiquitin Ligase CBL Confers Chemoresistance in Human Pancreatic Cancer and is Targeted by Epidermal Growth Factor Receptor Inhibition
Figure Lengend Snippet: CBL expression in human pancreatic ductal adenocarcinoma (PDAC) is inversely correlated with EGFR expression and activation. A, immunohistochemistry staining of human PDAC tumors stained for CBL, pEGFR, and EGFR reveals an inverse expression in 12 of 15 tumors (2 representative individual patient samples are shown), suggesting a possible regulatory effect of CBL on EGFR. B, correspondingly, when stimulated by EGF ligand, immunoblots of Panc-1 cells with CBL knockdown show increased phosphorylation of EGFR (Y1068) when compared to the isogenic parental cell line, with increased downstream pERK and pAKT (columns 2 versus 4). Relative densitometry values normalized to β-actin as a loading control are displayed above each phosphoprotein band and, C, plotted as a bar graph.
Article Snippet: Membranes were first blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline + 0.1% Tween20 (TBST), washed ×3 in TBST, then incubated overnight at 4°C in 5% BSA in TBS containing primary antibody at 1:1000 dilution unless otherwise noted: CBL, pAKT 1:500, AKT, pEGFR (Y1068), pEGFR (Y1045), EGFR 1:2000, pERK, ERK (Cell Signaling). β-actin 1:5000 (Sigma) was used to normalize sample loading.
Techniques: Expressing, Activation Assay, Immunohistochemistry, Staining, Western Blot