Fluorchem hd2 system

Following the various treatments, cells were lysed in a solution containing the following: 20 mM Tris at pH 8, 150 mM NaCl, 1% triton, 0.1% SDS, 2 mM sodium orthovanadate, 2 mM PMSF, 50 mM NaF, and protease inhibitors (Roche). For tissue samples, cryosections were collected, and the sections placed into the above lysis buffer and homogenized using a Tissuelyzer device (Qiagen). Lysates from cells or tissues were clarified with centrifugation, normalized for protein concentration and separated on Nupage Novex 10% Bis-Tris gels (Lifetech). Samples were then transferred using the iBlot System to a nitrocellulose membrane using the iBlot Gel Transfer Stacks (Life Technologies). The membrane was washed twice in phosphate buffered saline with 0.1% tween (PBST). The membranes were then incubated at 4°C overnight in a 1:500 dilution of glypican-1 antibody (Abcam), phospho-ERK, ERK, phosphor-AKT or AKT antibodies (Cell Signaling). Membranes were washed twice in PBST and incubated with an appropriate HRP-linked secondary antibody at 1:5000 dilution for two hours at room temperature. The membranes were washed extensively in PBST and imaged using the Femto Maximum Sensitivity Substrate (Thermo Scientific) with a FluorChem HD2 System (Cell Biosciences). Silver staining was performed on gels using a silver staining kit according to the manufacturer’s instructions (Thermo Scientific).