Enhanced chemi luminescence (ecl)

Cells were washed twice with ice-cold PBS and lysed in 200 μL of ice-cold RIPA buffer containing 150 mmol/L NaCl, 1% Nonidet P-40, 0.25% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mmol/L Tris (pH 7.4), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L Na₃VO₄, and 1 mmol/L NaF. The cell lysate was harvested and centrifuged at 10,000 × g and 4°C for 20 min. The supernatants were collected to new tubes, and the protein concentration of supernatants was determined using Bradford method. The total protein (30 μg) was then mixed with 6× loading buffer and boiled for 5 min. The sample mixture was run on 15% SDS-PAGE gels in 1× running buffer at 25 mA for 2 h. The protein was transferred to polyvinylidene difluoride membranes (PVDF; Millipore Corporation) at 70 V for 3.5 h in transfer buffer. Then, the membrane was blocked in TBST (0.5% Tween 20 in Tris-buffered saline) containing 5% (w/v) non-fat dry milk at 37°C for 1.5 h, and washed 3 times with TBST for 30 min. The membranes were incubated with respective primary antibodies for 16–18 h at 4°C. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody, and the bands were visualized with ECL (Promega) as described by the manufacturer. The intensity of bands was quantitated by Quantity-One software.

Co‐immunoprecipitation experiments were performed with Protein A/G PLUS‐Agarose (Santa Cruz Biotechnology) following the manufacturer's instructions. Briefly, to minimize non‐specific binding, 200 μg of protein lysates from treated neurons was prECLeared using a control agarose resin. The prECLeared lysates were incubated with 1 μg primary anti‐ubiquitin antibody for 1 hr at 4°C, and then 20 μl of resuspended volume of Protein A/G PLUS‐Agarose was added and incubated overnight. The resin was separated by centrifugation, washed three times with ice‐cold lysis buffer and then boiled in SDS sample buffer. Immunoblot analysis was performed with the primary antibody anti‐MAD2B, horseradish peroxidase‐conjugated antibody to mouse (1:20,000 dilution; Promega, Madison, WI, USA) and an enhanced chemiluminescence system (ECL, Amersham).

Briefly, total protein was extracted from the cells its concentration was determined by Lowry method. Total protein was separated by SDS-PAGE and then transferred to a PVDF membrane at 320 mA for 2 h at 4°C. The protein in the PVDF membrane was blocked with 5% fat-free milk in PBST, cultured in primary antibodies and then incubated in the secondary antibodies for about 1–2 h at room temperature. PVDF membranes were washed with PBST (3 × 10 min). The protein bands were visualized using ECL according to the the manufacturer's instructions (Promega).

Cells were treated with RIPA lysis buffer containing phenylmethyl sulfonylfluoride (PMSF). Protein concentration was measured using BCA Protein Assay Reagent (Pierce, US). Proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membranes (Millipore, US). The PVDF membrane was blocked with 5% non-fat milk for 2 h at room temperature and then subsequently incubated with primary antibodies overnight at 4 °C and HRP-conjugated secondary antibody at room temperature for 1 h. Finally, the membrane was developed with enhanced chemiluminescence (ECL) (Promega, US).

Cells were lysed with EZ-RIPA lysis buffer (ATTO, Tokyo, Japan) supplemented with a protease inhibitor (ATTO) and a phosphatase inhibitor (ATTO). Cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, proteins were transferred to Nitrocellulose membrane. Membranes were blocked for 1 h in Tris-buffered saline (TBS) containing 5% Bovine Serum Albumin (BSA) and 0.1% Tween 20 (TBS-T) and subsequently were incubated with the primary antibody overnight at 4 °C. The membranes were washed with TBS-T and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h. Blots were visualized by ECL (Promega, Madison, WI, USA) and Luminescent Image Analysis System.

Total cell lysates were prepared and analysed by western blot as previously described20 (link). The specific primary antibodies were used to detect the markers at 4 °C overnight. The blots were visualised using ECL (Promega, Madison, WI, USA) on a Kodak Image Station 4000 mm Pro System (Kodak, Rochester, NY, USA). The density of the bands was quantified by densitometric analysis using the Image Tool (version 3.0) system.