Protease inhibitor and phosphatase inhibitor cocktails 1 and 2

Total proteins were extracted from the cells using a hypotonic lysis buffer (50 mM Tris-HCl, pH 7.5, 1% NP40, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1 mM EDTA) containing protease inhibitor and phosphatase inhibitor cocktails I and II (Sigma). After 20-min centrifugation at 14,000 g at 4°C, the supernatant was collected and protein concentrations were measured using BioRad Protein Assay (BioRad). Secreted proteins were collected after a 72-h incubation by Amicon Ultra-15 Centrifugal Filter Units (Merck) according to manufacturer’s protocol. Proteins were separated by 10% or 12% SDS-PAGE and then electroblotted onto a Protran nitrocellulose transfer membrane (Schleicher & Schuell). Immunoblots were incubated with primary and secondary antibodies, and signal was enhanced using chemiluminescence (SuperSignal West Pico chemiluminescent substrate; Thermo Scientific). The signal was detected by exposure to X-ray film or using the Fusion FX7 image acquisition system (Vilber Lourmat). Data were quantified using ImageJ [66 (link)], or using the FusionCapt software. Reported are data from at least three separate experiments.

Total proteins were extracted from the cells using a hypotonic lysis buffer (50 mM Tris-HCl, pH 7.5, 1% NP40, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1 mM EDTA) containing protease inhibitor and phosphatase inhibitor cocktails I and II (Sigma). After 20 min centrifugation at 14,000 g at 4°C, the supernatant was collected and protein concentrations were measured using BioRad Protein Assay. Cytoplasmic and nuclear fractions were obtained using sucrose gradients as described [81 (link)]. Proteins were separated in an 8% SDS-PAGE and then electroblotted onto a Protran nitrocellulose transfer membrane (Schleicher & Schuell). Immunoblots were incubated with primary and secondary antibodies and visualized by enhanced chemiluminescence (SuperSignal West Pico chemiluminescent substrate; Thermo Scientific) and exposure to X-ray film. Data was from three separate experiments and was quantified using Image J. Error bars represent SEM.