Protease inhibitory cocktail

Transfected HEK cells or primary cultures of cortical neurons at 15 or 22-23 days in vitro (DIV) were washed 3 times in ice-cold PBS supplemented with 0.8 mM CaCl2 and 0.5 mM MgCl2 (PBS²⁺) and then incubated for 12 min at room temperature followed by further 12 min at 4°C with 1 mg/ml Sulfo-NHS-SS-Biotin (ThermoFisher Scientific) in PBS²⁺. After rinsing in ice-cold PBS²⁺, biotin was quenched in 50 mM glycine in PBS²⁺ for 10 min. Cells were scraped in NaCl-Tris buffer supplemented with protease inhibitory cocktail (Roche) and then lysed (150 mM NaCl, 50 mM TrisHCl, 2% Triton X-100, 2 mM EDTA, 1 mM PMSF, protease inhibitor cocktail) for 1 h at 4°C. Biotinylated proteins were pulled down by incubating cell lysates with neutravidin agarose beads (ThermoFisher Scientific) for 2 h at 4°C. After extensive washes, beads were resuspended in gel loading buffer (Sigma-Aldrich) and bound proteins were eluted with boiling. Relative cell surface expression levels were analyzed by western blotting. Inputs correspond to 20% of the cell surface fraction.

Cells were harvested and washed twice with PBS. Cell lysis was performed on ice for 25 min, in RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl, pH 8.0) containing a protease inhibitory cocktail (Roche). Insoluble material was pelleted by centrifugation at 16,000× g at 4°C for 3 min. Protein concentrations were determined using the Bradford assay (Bio-Rad). Thirty micrograms extract was mixed with 4× Laemmli buffer (40% glycerol, 240 mM Tris/HCl, pH 6.8, 8% SDS, 0.04% bromophenol blue, 5% β-mercaptoethanol), denatured at 96ºC for 5 minutes, separated by SDS-PAGE, and transferred to nitrocellulose membranes (PROTRAN-Whatman, Schleicher&Schuell). The membranes were blocked with 5% non-fat dry milk in TBS-T for 60 min, incubated with primary antibodies overnight at 4°C, washed three times with TBS-T for 10 min, incubated with the peroxidase-conjugated secondary antibody (1:2000; Amersham Biosciences) in TBS-T with 5% non-fat dry milk for 60 min, and washed three times with TBST for 10 min. Immunoreactive proteins were detected using Supersignal West Dura HRP Detection kits (Pierce). The primary antibodies used were: p16^Ink4a (Santa Cruz); p19^Arf (Ab80 Abcam); p15^Ink4b (Santa Cruz); β-catenin (clone 14, BD Biosciences); p53 (sc-6243 Santa Cruz); p21 (BD Pharmigen); c-Myc (sc-764 Santa Cruz); β-actin (ab8226, abcam).