Anti gapdh antibody 6c5

Cells were harvested and lysed in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS/PAGE) sample buffer (50 mm Tris/HCl, pH 6.8, 2% SDS, and 10% glycerol). Protein concentration was determined by Bio‐Rad DC protein assay (Bio‐Rad). Proteins were separated by SDS/PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore, Carrigtwohill, Ireland). The membrane was blocked with 1% skimmed milk in Tris‐buffered saline containing 0.05% Tween 20 and probed with appropriate antibodies using the Envision system (Dako, Glostrup, Denmark). Signals were developed by Luminata Forte HRP substrate (Millipore) or SuperSignal West Femto Substrate (Thermo Fisher Scientific, Rockford, IL, USA) and then captured by ChemiDoc MP Imaging System (Bio‐Rad). Following primary antibodies were used: anti‐p53 antibody (FL393; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐p21 antibody (H164; Santa Cruz Biotechnology), anti‐CES2 (G5; Santa Cruz Biotechnology), and anti‐GAPDH antibody (6C5; Santa Cruz Biotechnology).

Mice were euthanized by decapitation and their hippocampi were dissected and stored at −80° C. Six hippocampi from TS and CO mice were lysed and homogenized as previously described [68 (link)]. The homogenates were boiled for 10 min and sonicated for 5 cycles of 30 s On/Off at 4° C using a Bioruptor Plus (Diadode) and left on ice for 20 min. The total protein content of each sample was determined following the protocol described by [88 (link)]. Identical amounts of protein from each sample were loaded on a 15% sodium dodecyl sulfate-polyacrylamide gel, electrophoresed and transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were incubated with a polyclonal rabbit anti-acetyl histone H4 (06-598, Upstate), polyclonal rabbit anti-tri-methylated histone H4 at K20 (07-463, Upstate) and polyclonal rabbit anti-nucleolin (ab22758, Abcam) overnight at 4° C. To ensure equal loading, the blots were reproved using a mouse monoclonal anti-GAPDH antibody (6C5) (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After extensive washing, immunoblots were developed with goat anti-rabbit IRDye 680RD antibody or goat anti-mouse IRDye800CW (1:10,000; LI-COR Biotechnology, Lincoln, NE, USA)
Protein bands were detected using a LI-COR ODYSSEY IR Imaging system V3.0 (LI-COR Biotechnology) and quantified following the protocol described by [68 (link)].

The purity of the recombinant MPP1 protein was checked using SDS–PAGE and Western blot analysis or Coomassie staining. Proteins were separated on 10% Tris-Glycine SDS–PAGE gel and electrotransferred on a nitrocellulose membrane (Amersham™ Protran^®) or fixed in 50% methanol. The membrane was blocked with 5% skim milk in TBS-T (20 mM Tris, pH 7.4; 150 mM NaCl, 0.05% Tween-20) overnight at 4 °C, then incubated with primary antibodies (Anti-His-tag Antibody (AD1.1.10) or Anti-GAPDH Antibody (6C5), Santa Cruz Biotechnology, Dallas, TX, USA) in TBS-T overnight at 4 °C with secondary antibodies conjugated with HRP (Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L), Jackson Immuno Research) in TBS-T for 1 h at room temperature. Between each incubation step, membranes were washed with TBS-T. The blots were developed with Radiance ECL (Azure Biosystems, Dublin, CA, USA) and imaged with the Azure image system (Azure 600; Azure Biosystems). The methanol-fixed gels were stained with Coomassie Brilliant Blue G-250, then washed with distilled water and imaged with the Azure image system.