Ab47915

Total proteins were isolated from vegetative hyphae harvested from 12 h YEPD cultures as described [24 (link)]. Acetylation of histone H3 and H4 was detected with anti-H3ac (ab47915), anti-H4ac (ab177790), and anti-H4K16ac (ab194352) antibodies from Abcam (Cambridge, UK). Detection with the anti-H3 (ab209023, Abcam), and anti-H4 (ab10158, Abcam) antibodies were used as the loading controls. The band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Relative intensity of the histone acetylation was normalized to that of non-acetylated histone, and compared with that of the control group [67 (link)]. Each experiment was performed for three independent biological replicates, and error bars represent standard deviation estimated with data from three independent replicates.

For Western blot analysis of the H3, H3K9, H3K14, H3K18, and H3K27 acetylation levels in C. neoformans, the indicated strains were cultured overnight at 30°C in 25 mL YPD liquid medium. Protein extraction and Western blotting were carried out as previously mentioned (56 (link)). Briefly, 1 mL precooled protein lysis buffer containing protease inhibitor (A32963, Thermo Fisher), 1 mM phenylmethylsulfonyl fluoride (PMSF, P8340, Solarbio Technology Co., Beijing), and 200 µL (~1 PCR tube) 1.0 mm zirconia beads were added to the collected cells. The cell was then broken five times at maximum speed for 40 s using a cell wall breaker (MiniBeadBeater-16, BioSpec) with 1-min interval on ice, followed by centrifuge at 12,000 rpm for 5 min at 4°C to get the protein supernatant. The BCA protein assay kit (A53225, Thermo Fisher) was used for protein quantification, and the quantified protein was further separated by SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membrane (ISEQ00010, Millipore). Immunoblotting was examined with specific antibodies against H3Ac (ab47915, Abcam), H3K9Ac (ab4441, Abcam), H3K14Ac (ab52946, Abcam), H3K18Ac (ab1191, Abcam), H3K27Ac (ab4729, Abcam), and H3 (4499S, Cell Signaling Technology), respectively.

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.

To detect trimethylated H3K27 after specific treatments, coverslips with cultured HPdLFs were fixed in 4% PFA for 10 min, washed with phosphate-buffered saline (PBS) and incubated with the primary antibody for 1.5 h and the secondary antibody for 45 min. DAPI (Thermo Fisher Scientific; 1:10,000 in PBS) was used for nucleus staining. The following antibodies were used: rabbit anti-human H3K9/14/18/23/27 (ab47915; Abcam, Cambridge, UK; 1:500), mouse anti-human H3K27me3 (ab6002; Abcam; 1:250), goat anti-rabbit Cy5 and goat anti-mouse Cy5 (111-175-144 and 115-175-146; Jackson ImmunoResearch, West Grove, PA, USA; 1:1000).

The tested seedling samples were ground, the total proteins were extracted, and specific proteins were detected as described [54 (link)]. Histone H3 was used as an equal loading control. The antibody in this study used in Western blotting was anti-H3K9K14K18K23K27ac (ab47915, Abcam, Lot: GR137984-20, Cambridge, UK).

Total cell extracts were prepared in lysis buffer containing 62.5 mM Tris–HCl, 2% (w/v) SDS and 10% sucrose (pH 8) supplemented with EDTA-free protease (Roche, 04693159001) and phosphatase inhibitor cocktails (Roche, 04906837001) and sonicated four times at 60 Hz for 10 s on ice. Fractionation experiments after overexpression of aggregation-prone proteins were performed as described earlier [56 (link)] and specified in the Supplemental Methods. For Western blotting, equal amounts of proteins were separated by SDS-PAGE under denaturing conditions. Proteins were transferred on nitrocellulose membrane and proteins were detected by specific antibodies recognizing SQSTM1 (Progen, GP62-C), LC3B (Novus, NB-100-2220), FLAG (Sigma, F1804), tubulin (Sigma, T9026), SOD1 (Epitomics, 2018-1), ATG9A (Abcam, ab108338), histone H3 (Abcam, ab47915), α-synuclein (Abcam, ab27766), and DNAJC13 (kind gift from M. Farrer, Vancouver, and P. McPherson, Montreal, Canada). Species-specific secondary antibodies coupled to horseradish peroxidase (Dianova) were used to develop the Western blot with the Fusion system (Peqlab) or the Amersham Imager 600 (GE). Densitometry was performed with the AIDA software (Raytest) or ImageJ.