Broad spectrum protease inhibitor cocktail

To detect ubiquitinated Bim in whole-cell extracts, granulosa cells were homogenized in RIPA buffer containing a broad-spectrum protease inhibitor cocktail (Roche, Basel, Switzerland). The protein concentrations of the cell extracts were measured, and equal amounts of protein were incubated overnight at 4 °C with a polyclonal Bim antibody (Cell Signaling Technology, Beverly, MA, USA). The antibody-antigen complex was then incubated with protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoprecipitated proteins were resolved by SDS-PAGE (12% acrylamide gel) and processed for Western blotting using specific antibodies to detect ubiquitin and Bim.

Human hippocampus was homogenized in 0.01 M phosphate buffer, pH 7.0, containing a broad spectrum protease inhibitor cocktail (Roche) using mechanical homogenization and three freeze–thaw cycles. Homogenates were centrifuged for 10 min at 12,000 rpm at 4 °C. Total protein levels in each sample were quantified using the bicinchoninic acid assay (Pierce) and 50 μg protein was loaded per lane of a 12 % SDS-PAGE mini-gel. After separation, the proteins were transferred onto a Hybond-ECL nitrocellulose membrane (GE Healthcare) using a Mini Trans-Blot Cell (Bio-Rad) and equal loading was confirmed with Ponceau-S (Sigma). Membranes were blocked for 1 h at room temperature in TBS containing 0.05 % Tween-20 (TBST) and 5 % skimmed milk and then probed with mouse anti-CYP26A1 antibody (1:1000 in blocking buffer; Vertebrate Antibodies) at 4 °C overnight. Membranes were washed in TBST and then incubated in HRP-conjugated anti-rabbit secondary antibody (1:3000; Jackson ImmunoResearch) for 1 h at room temperature. The membranes were washed again in TBST and antibody binding visualized by enhanced chemiluminescence (ECL; Millipore) and exposure to X-ray film (Thermo).

Cells were washed twice with PBS and lysed in RIPA lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% Nonidet P-40) supplemented with a broad-spectrum protease inhibitor cocktail (Roche). Protein concentrations were measured using the Bradford assay. Proteins were denatured by boiling for 5 min. Samples were resolved by 10% SDS-PAGE and transferred to Hybond ECL membranes (Amersham Pharmacia Biotech). The membranes were blocked for 30 min in Tris-buffered saline containing 0.1% Tween 20 (TBS/T) and 5% non-fat milk, and then incubated overnight at 4°C with primary antibodies against LC3 (Sigma-Aldrich), IFT88 (ProteinTech Group), Arl13B (ProteinTech Group), Polyglutamylation Modification (GT335, AdipoGen), and GAPDH (Abcam). The membranes were washed three times with TBS/T and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Phototope-HRP Western blot detection Kit; New England Biolabs) for 2 hr at room temperature. After three washes for 10 min each, the blots were developed using the LumiGLO chemiluminescent substrate (Cell Signaling Technology).