Rabbit anti mouse secondary antibody

Forty μg of lung tissue lysate proteins, as determined by a Bradford protein assay were loaded equally onto 10% polyacrylamide gels (Life Technologies Corporation; Carlsbad, CA), and transferred to PVDF membranes (Life Technologies, Corp.). Membranes were blocked for one hour at room temperature in 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST), and then probed with anti-mouse phospho (p)-SHP-1 (Tyr564), p-SHP-2 (Tyr580), p-Akt (Thr308) or SHP-1, SHP-2, Akt antibodies (Cell Signaling Technology, Inc., Danvers, MA) overnight at 4°C in TBST containing 5% bovine serum albumin (BSA). After primary antibody incubation, membranes were washed three times in TBST and rabbit anti-mouse secondary antibody (Cell Signaling Technology, Inc.) was added at a concentration of 1:3000 in TBST with 5% BSA. β-actin was blotted as a loading control. Membranes were developed by chemiluminescence using an Amersham prime ECL plus detection system (GE Healthcare Life Sciences; Pittsburgh, PA) and densitometric analyses were performed as previously reported by our laboratory ^{[21 (link)]}.