Ab45152

Hyphae were harvested from 24 h YEPD (Yeast extract peptone dextrose) cultures by filtration through two layers of Miracloth (Sigma, USA) and washed with sterile distilled water. Proteins were isolated from vegetative hyphae as described [63 (link)]. For Western blot analyses, total proteins were separated on 12.5% SDS-PAGE gels and transferred to nitrocellulose membranes. Acetylation of histone H3 and H4 was detected with the anti-Histone H3ac (K9+K14+K18+K23+K27) (ab47915), anti-Histone H4ac (K5+K8+K12+K16) (ab177790), anti-Histone H4K5ac (ab51997), anti-Histone H4K8ac (ab15823), anti-Histone H4K12ac (ab46983), anti-Histone H4K16ac (ab194352), and anti-Histone H2AK5ac (ab45152) antibodies from Abcam (Cambridge, UK). Detection with the anti-Histone H3 (ab209023, Abcam), anti-Histone H4 (ab10158, Abcam), and anti-Histone H2A (ab188312, Abcam) antibodies was used as the loading controls.

IF staining was performed according to a previous study (Su et al. 2011 (link)). Samples were fixed with 4% paraformaldehyde and rinsed three times, 5 min each, with PBS-PVA (PBS containing 0.2% PVA) and permeabilized with 0.1% Triton X-100 for 30 min at room temperature. Samples were incubated overnight with anti-H2A type 2-C (1:1000; ab45152, Abcam), anti-H2B type 1 (1:1000; ab177430, Abcam) or anti-CDX2 (1:100; EPR2764Y, Invitrogen) at 4°C. Afterward, the samples were rinsed three times for 5 min each with PBS-PVA, incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:500; ab150077, Abcam) or goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor™ Plus 555 (1:500; A32727, Invitrogen) for 2 h at room temperature in the dark and rinsed with PBS-PVA. DAPI (1:500; C1005, Beyotime) nuclear stain was added for 8 min at room temperature in the dark, and rinsed three times with PBS-PVA. The specific signals were observed and imaged using a laser scanning confocal microscope (Carl Zeiss, Germany) under the same conditions. Fluorescence signal intensities were measured according to a previous report (Shi et al. 2023 (link)).

Western blotting was carried out following a previous study from our laboratory (Han et al. 2018 (link)). GFFs transfected with pcDNA3.1(+)-lncRNA3720 were transferred into lysis buffer and total protein was extracted. Protein aliquots were separated by SDS–PAGE on a 5% stacking gel, and a 10% separating gel at 120 V for 1.5 h and then electrophoretically transferred onto a polyvinylidene fluoride membrane at 250 mA for 2.5 h. Membranes were blocked in TBST (Tris-buffered saline pH 7.4 with 0.1% Tween 20) containing 3% BSA for 2 h at room temperature and then incubated overnight with anti-H2A type 2-C (1:1000; ab45152, Abcam) or anti-H2B type 1 (1:500; ab177430, Abcam) or anti-β-actin (1:500; HC201-01, Trans) at 4°C. After washing three times with TBST for 10 min each time, the membrane was incubated for 2 h at room temperature with horseradish peroxidase-labeled goat anti-rabbit IgG(H+L) (1:1000; A0208, Beyotime). After washing three times with TBST, 10 min each time, the signals were detected using ECL (Beyotime). The intensity values of the different protein bands were measured by Image J (Media Cybernetics, Silver Spring).