Histone Acetylation Western Blots

Western blotting analyses were performed on four samples of purified histones, given that the quantity of histones isolated from the other two nuclear protein extracts resulted not sufficient. For the analysis, we followed the instructions of the Acetyl Histone Antibody Sampler Kit (Cell Signaling) and the protocol applied by Mátis et al. [7]. Before using “Acetyl-Histone H4 (Lys8) Antibody #2594”and Histone H4 (L64C1) #2935 provided with the kit, we used ClustalW to perform a multiple sequence alignment between the human histone H4 peptide sequence that was used for the production of antibodies and the ortholog sequences in European seabass (Dicentrarchus labrax), and other teleosts such as zebrafish (Danio rerio), Nile tilapia (Oreochromis niloticus), and Atlantic salmon (Salmo salar). As shown in S1 Fig, the histone H4 peptide sequence in European sea bass presents 100% similarity with the human sequence, and it is the same for the other teleosts’ histone H4 sequences. This suggest that antibodies were suitable for the detection of the antigen in our target species.

Histone proteins were diluted by 2x SDS and β -mercaptoethanol containing loading buffer (supplemented with 50 mM DTT), sonicated for 15 s, and heat denatured at 95°C for 5 min. Histones were separated by SDS-PAGE on polyacrylamide (4–20%) precast gradient gels (Bio-Rad); 3 μg histone protein per lane were loaded for the detection of histones H2A, H2B, and H3, whereas 6 μg per lane were loaded for histone H4. After electrophoresis, proteins were blotted onto PVDF membranes (0.22-μm pore size, Bio-Rad). Before proceeding to the immunodetection process, a reversible Ponceau staining was applied to membranes to test equal loading of gels and protein transfer. Histones were identified using antibodies furnished by the Acetyl Histone Antibody Sampler Kit. After blocking with 5% fat-free milk containing PBST for 3 h, the immunoblots were incubated overnight at 4°C with primary antibodies against histone H2A (1:1000), H2B (1:500), H3 (1:1000), H4 (1:500), and their acetylated forms. Each acetyl histone antibody was specific for the target histone modified at the lysine residue of the most frequent acetylation site (AcH2A and AcH2B: Lys 5, AcH3: Lys 9, AcH4: Lys 8). The primary antibody was detected using an anti-rabbit secondary antibody (1:2000) or an anti-mouse secondary antibody (1:900) for the non-acetylated H4 histone. Both secondary antibodies were coupled with horseradish peroxidase. Primary antibodies were diluted in PBST containing 5% BSA, with the exception of anti H4, which was diluted in PBST containing 5% of defatted milk. Secondary antibodies were diluted in PBST containing 5% fat-free milk. Signals were detected using an enhanced chemiluminescence system (SuperSignal®west Dura Extended Duration Substrate, Thermo Scientific) and exposing to clear-blue X-ray film. After film exposure, densitometry was used to quantify protein levels on the western blots by means of Quantity One 1-D software (Bio-Rad). The protein levels were expressed as adjusted volume, Adj. Vol. [OD*mm²] = [{Sum of the intensities of the pixels inside the volume boundary} x {area of a single pixel in mm²}]–{the background volume}).