Ecl plus substrate

The amounts of viral vectors were normalized to the amount of HIV p24 (1 mg/ml) and mixed with LDS sample buffer (ThermoFisher Scientific) with 2-mercaptoethanol. Each sample (20 µl) was subjected to electrophoresis through an SDS 12% polyacrylamide gel (ThermoFisher Scientific). Immunoblot analyses of envelope proteins were performed with: 1) rabbit anti-Sindbis virus polyclonal antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal antibody (ThermoFisher Scientific); 2) HRP-conjugated rabbit goat anti-goat immunoglobulin antibody (ThermoFisher Scientific); and 3) biotinylated HRP (ThermoFisher Scientific). The protein bands were visualized by ECL plus substrate (ThermoFisher Scientific) and ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA).

The generation and purification of the rabbit polyclonal antibody specific for p53 dimethylated at K382 was described previously (Kachirskaia et al., 2008 (link)). The anti-p53K381ac antibody was obtained from Abcam (ab61241, Abcam, Cambridge, MA). Peptides were dissolved in water to yield 1 or 2 mg/mL stock solutions. The peptides were further diluted into 0.1% BSA in PBS. One microliter of the peptide dilutions were spotted onto a Whatman Protran nitrocellulose membrane (0.45 µm pore size, GE Healthcare Life Sciences, Pittsburgh, PA), allowed to air dry for 1h at room temperature and stored at 4 °C overn ight. Membranes were blocked with 1% BSA in PBS-T (PBS with 0.1% Tween-20) for 1h, incubated with a 1:100 dilution of antip53K382me2 for 1h at 4 °C, then washed 5 times with PBS-T. The membranes were incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Thermo Scientific, Rockford, IL) for 1h at room temperature with shaking, then washed 5 times with PBS-T. Images were developed with ECL Plus substrate (Thermo Scientific, Rockford, IL) and exposed to Hyperfilm ECL (GE Healthcare Life Sciences, Pittsburgh, PA) or imaged using a ChemiDoc MP imaging system (BioRad).

Western blot assay is performed for protein expression. Briefly, protein was extracted using 1x RIPA buffer. Equal amount of proteins from brain were separated by SDS-PAGE. Further, protein from the gel was transferred onto PVDF membrane (BioRad, Hercules, CA) by wet transfer method. Non-specific sites of protein on membrane were blocked with 5% non-fat dry milk in TBS-T (50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween- 20, pH 7.4) for 1 h at room temperature. Membranes were washed with washing buffer (pH 7.6, TBS, 0.1% Tween 20) for 3 times, 10 min each. The blot was then incubated for overnight at 4°C with appropriate primary antibody in blocking solution according to the supplier’s specific instructions. The blots were washed with TBS-T (3 times, 10 min each) and incubated with appropriate HRP- conjugated secondary antibody for 2 h at room temperature. After washing with TBST-T, ECL Plus substrate (Thermo scientific, inc.) was as applied to the blot for protein expression and images were capture in gel documentation system. Relative optical density of protein bands was analyzed by using software image lab 3.0. The same membranes were used for estimation of GAPDH protein which was stripped with striping buffer and re-probed with GAPDH. The GAPDH was used as a loading control for all the proteins of interest.