Halt phosphatase inhibitor mixture

ECM-detached cells were harvested, washed once with cold PBS, and lysed in 1% Nonidet P-40 supplemented with protease inhibitors leupeptin (5 µg/mL), aprotinin (1 µg/mL), and PMSF (1 mM) and the Halt Phosphatase Inhibitor Mixture (Thermo Scientific, Waltham, MA, USA). Lysates were collected after spinning for 30 min at 4 °C at 14,000 rpm and normalized by BCA Assay (Pierce Biotechnology, Waltham, MA, USA). Normalized lysates underwent SDS-PAGE and transfer/blotting was performed as previously described^{16 (link)}. Membranes were cut prior to incubation with primary antibodies when blotting for more than one target at a time. The following antibodies were used for western blotting: FLIP (Cell Signaling Technology, #56343), phospho-Akt (Ser473) (Cell Signaling Technology, #4060), GAPDH (Cell Signaling Technologies, #5174), β-tubulin (Cell Signaling Technology, #2146), β-Actin (Sigma-Aldrich, #A1978), phospho-IκBα (Ser32/36) (Cell Signaling Technology, 9246s), phospho-GSK-3β (Ser9) (Cell Signaling Technology, 5558s), and c-myc (sigma M-5546). Original, unprocessed data are available in Supplemental Figs. 5, 6.

Spleens were harvested from all experimental groups, homogenized, and made up the cell lysate. Whole cell lysate was prepared by using lysis buffer (50 mM Tris-HCl [pH 7.4], 5 mM EDTA, 120 mM NaCl, 0.5% Nonidet P-40, 0.5 mM NaF, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) along with HALT phosphatase inhibitor mixture (78420, Thermo Fisher Scientific) and protease inhibitor mixture (78410, Thermo Fisher Scientific) for 1 hour. Samples were electrophoresed on a 10% SDS-polyacrylamide gel and electroblotted onto polyvinylidene difluoride membranes. Blots were blocked for 1 hour in 5% BSA in PBS with 0.1% Tween 20. NF-κB, pNF-κB, FOXO1, and pFOXO1 proteins were detected with NF-κB (8242S), pNF-κB (3033S), FOXO1 (2880S), and pFOXO1 (9461S) monoclonal antibodies, respectively, at a dilution of 1:250 and as recommended by the manufacturer (Cell Signaling Technology). Goat anti–rabbit IgG G-conjugated horseradish peroxidase (sc-2004) (diluted 1:5000) was used as a secondary antibody (Santa Cruz Biotechnology Inc.). Immunoblotting for β-actin was carried out to confirm equal loading.