Ecl advance western blotting detection kit

To collect protein for immunoblotting, WT neurons were cultured at high density (~200,000 neurons per well) in a 48-well plate. Four days after plating, cells were stimulated with 500 μM AraC for 15, 30 and 60 min.
Protein samples were prepared for SDS-PAGE in SDS sample buffer (Merck Millipore; 70607) and boiled at 95 °C for 10 min before electrophoresis on 12% gels. Proteins were transferred to PVDF membranes (Amersham, GE10600023). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies.
The following primary antibodies were used at the indicated dilutions: rabbit anti-phospho Y515 TrkB (Abcam: ab131483; 1:500), goat anti-TrkB (R&D systems; AF1494; 1:500), rabbit anti-IκBα (Santa Cruz; 9165; 1:500), rabbit anti-phospho-c-Jun (Thr91, Cell Signalling Technology; 2303; 1:1000), rabbit anti-c-Jun (Cell Signalling Technology; 9165; 1:1000) and mouse anti-GAPDH (Sigma; G8795; 1:1000). Immunoreactivity was visualised using appropriate HRP-conjugated secondary antibodies (Cell Signalling Technology; 7074). Immunoblots were developed using the ECL Advance Western blotting detection kit (Thermo Scientific; 34095) and imaged using a chemiluminescent western blot imaging system, Azure c300 (Azure Biosystems). Image analysis and quantification of band intensities were done using NIH ImageJ software.

Total mitochondrial proteins were resolved on 4–15% Tris-glycine SDS-PAGE gels and electroblotted onto polyvinylidene fluoride membranes (Bio-Rad, Marnes La Coquette, France). Following electrotransfer, membranes were blocked for 1 h at room temperature in 5% BSA-PBST (10 mM Tris-HCl, pH 8.0/150 mM NaCl/0.1% Tween-20). Next, membranes were incubated overnight at 4 °C with primary antibody. The day after, the membranes were washed six times with PBST and incubated with peroxidase-conjugated secondary antibody at room temperature for 1 h. Peroxidase activity was detected with enhanced chemiluminescence (ECL Advance Western Blotting Detection Kit; Thermo Scientific, Villebon sur Yvette, France). For protein detection, the following antibodies were used: sAC (Abcam Cambridge, UK; CEP Biotech, Tamarac, FL, USA), Epac1, 2 (Cell Signaling, Danvers, MA, USA), ANT (Abcam), GAPDH (Cell Signaling), VDAC (Genosphere, Paris, France), TnI (Cell Signaling), PLB (Cell Signaling) and MCU (Biorbyt, Berkeley, CA, USA).

Immunoblotting protein samples were prepared for SDS-PAGE in SDS sample buffer (Life Technologies) and boiled at 95 °C for 10 min before electrophoresis on 12% gels. Proteins were transferred to PVDF membranes (Amersham). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies. The following primary antibodies were used at the indicated dilutions: rabbit phospho-c-Jun (Thr91) (Cell signaling, 2303, 1:1000), rabbit anti-c-Jun (Cell signaling, 9165, 1:1000) and rabbit anti-GAPDH (Sigma, G9545, 1:1000). Immunoreactivity was visualised using appropriate HRP-conjugated secondary antibodies. Immunoblots were developed using the ECL Advance Western blotting detection kit (Life Technologies) and exposed to Kodak X-Omat AR films. Image analysis and quantification of band intensities was done with ImageQuant (GE Healthcare).