Super rx x ray film

Protein concentrations were measured with the BCA assay (Pierce). For immunoprecipitation analyses, only the input samples were measured. All protein samples were analyzed with SDS-PAGE, using either 8 or 12% polyacrylamide gels or 4–15% gradient gels (Bio-Rad). For whole cell lysates, 10–30 µg was loaded, while for immunoprecipitates 30 µg input material and the corresponding volume of flow-through material were applied to the gels. Separated proteins were then electroblotted to nitrocellulose membranes (0.45 µm, Bio-Rad), which were subsequently blocked with 5% (w/v) dry milk in PBS and probed with the primary antibody of interest diluted in PBS containing 1% (w/v) bovine serum albumin (BSA), 0.05% Tween 20 and 0.025% azide at 4 °C, overnight. Membranes were washed in PBS and PBS-Tween20 (0.05%) prior to 1 h of incubation at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. Following repeated washing steps, membranes were revealed by enhanced chemiluminescence (GE Healthcare Biosciences) and exposed to SuperRX X-ray films (Fujifilm Corporation).

The mouse forebrains were homogenized in DPBS containing protease and phosphatase inhibitors, followed by lysis in 2× RIPA lysis buffer. The neurons were directly lysed in 1 × RIPA buffer containing protease and phosphatase inhibitors. The protein lysates were centrifuged at 10,000×g at 4 °C for 5 min. The supernatants were saved, and the protein concentration was determined. Equal amounts of protein from each sample were run onto an SDS-PAGE gel, and the separated proteins were transferred to a nitrocellulose membrane. The membrane was blocked with blocking buffer (5% milk in TBS-T) at room temperature for 1 h, followed by incubation with primary antibodies including Cdk5 (1:1000), p35 (1:1000), p39 (1:2000), WDFY1 (1:2000), α-Tubulin (1:10,000), and actin (1:10,000) overnight at 4 °C. The membrane was subsequently incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Signal detection was performed using SuperSignal WestPico Chemiluminescent Reagents and exposed to a Fujifilm Super RX X-ray film. The films were developed by a Kodak medical X-ray film processor.

Protein was isolated by direct lysis of cells grown in culture using RadioImmuno-Precipitation Assay (RIPA) buffer (15 mM HEPES, pH 7.4, 2% Triton X-100, 145 mM NaCl, 0.1 mM MgCl₂, 10 mM EGTA, 1 mM sodium orthovanadate, 1 mM PMSF, aprotinin) supplemented with Complete protease inhibitors (Complete Mini-EDTA Free, Roche, Mannheim, Germany). Protein concentrations were determined using a BCA assay. Immunoprecipitation was performed using 4 μg antibody and Protein G Sepharose using the Immunoprecipitation Starter Kit and the manufacturer's instructions (GE Healthcare, Rydalmere, NSW). Proteins were separated using 10% SDS–PAGE gels then transferred onto BioTrace™ NT nitrocellulose membrane (Pall, Pensacola, FL). Membranes were blocked with Western blocking reagent (Roche) before incubation with primary antibodies to EphB4 including H200 (1:1000) and Zymed (1:1000) and to actin (1:10,000). Immunoreactivity was detected using the Amersham™ ECL Plus Chemiluminescence kit (GE Healthcare) and exposed to SuperRX X-ray film (Fuji Film Corporation, Japan).

Cells were lysed in SDS-loading buffer [50 mM Tris (pH 6.8), 2% (v/v) SDS, 10% glycerol, 5 mg/ml bromophenol blue] for 20 min on ice, briefly sonified and incubated at 45°C for 20 min. For reducing conditions, 4% (v/v) β-mercaptoethanol was added to the lysis buffer. Lysates were cleared by centrifugation and run on 10% SDS-polyacrylamide gels in a Mini-PROTEAN 3 cell apparatus (Biorad). Proteins were electroblotted onto Immobilon P membranes (Millipore) using a Bio-Rad Mini trans-blot cell apparatus at 100 V for 60 min at 4°C. The blots were probed with anti-PTH1R antibodies (1:4,000), followed by horseradish peroxidase-conjugated goat anti-rabbit (1:10,000) and detection on Super RX X-ray film (Fujifilm) using ECL Plus reagent (GE Healthcare).