Goat anti mouse antibody conjugated with horseradish peroxidase

For phospho-PKA substrate Western blots, protein extracts were prepared from trichloroacetic acid -treated cells. Cells were collected and suspended in 250 μl of 10% trichloroacetic acid. Next, the cells were disrupted with glass beads on Precellys® 24 homogenizer (Bertin Technologies). After that, the trichloroacetic acid pellets were resuspended in 100 μl of 2× SDS gel-loading buffer plus 50 μl of 1 M Tris base to adjust the pH. Samples were then boiled for 10 min and followed by centrifugation at 14,000×g for 5 min. The supernatants were separated on 8% SDS-PAGE gels and transferred onto nitrocellulose membranes. Blots were then blocked with 5% bovine serum albumin (BSA) in TBS/0.1% Tween 20. For phospho-PKA substrate, blots were probed with phospho-PKA substrate (RRXS*/T*) antibody (1:1000; cat no. 9624, cell signaling technology) followed by goat anti-rabbit antibody conjugated with horseradish peroxidase (1:5000; Santa-Cruz). For β-actin, blots were probed with anti-beta actin antibody (1:5000; cat no. ab8224, Abcam) followed by goat anti-mouse antibody conjugated with horseradish peroxidase (1:5000; Santa-Cruz). Finally, the blots were developed with ECL prime Western blotting detection reagent (Amersham Pharmacia Biotech).

N27 cells and brain tissue lysates were obtained using RIPA buffer (50 mM Tris, pH = 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and a cocktail of protease inhibitors) and mechanical dissociation. Lysates were then centrifuged, and the supernatant was collected and quantified using a Pierce bicinchoninic acid protein assay Kit (Thermo Scientific) according to the manufacturer’s instructions. Then, 40 µg of lysate protein was loaded in a 10% SDS polyacrylamide gel (at 110 V until the samples reached the end of the gel). After electrophoresis and transfer into a polyvinylidene difluoride membrane (1.0 A, 25 V for 25 min at room temperature, using a Trans-blot Turbo System (Bio-Rad)), specific protein bands were detected by using appropriate primary antibodies (mouse anti-CtBP1 and mouse anti-CtBP2, 1:2500; mouse anti-GAPDH, 1:5000, Millipore) and secondary antibodies (goat anti-mouse antibody conjugated with horseradish peroxidase, 1:5000, Santa Cruz Biotechnology) followed by incubation with Luminata Crescendo Western HRP Substrate (Millipore) for 5 min. Protein bands were detected using the ChemiDocTM MP Imaging System (Bio-Rad) and quantified by densitometry analyses using the Image Lab 5.1 software (Bio-Rad Laboratories).