P8340

In brief, cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% NP-40; 0.25% NaDoc; 10% Glycerol) containing phenylmethylsulfonyl fluoride (PMSF; Solarbio, P8340) and protease inhibitor cocktail (1:100, P8340; Sigma-Aldrich) for 15 min at 4 °C. 5% of total lysates were used as input for each sample. The remaining lysate was incubated with 1 μg of primary antibody on the rotator at 4 °C overnight. Protein G sepharose was then added and incubated for another 4 h at 4 °C. Protein G sepharose-enriched complexes were resolved on SDS-PAGE gels and transferred onto PVDF membranes.
Immunoblotting of the cell lysates and immunoprecipitates was performed using primary antibodies as indicated overnight at 4 °C and then with secondary antibodies for 1 h at room temperature. The bands were detected and visualized using a Hypersensitive ECL Chemiluminescence Kit (ABP Biosciences, Beltsville, MD).

The N. benthamiana leaves were ground in liquid nitrogen and homogenized in extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.2% Triton X-100 (Solarbio, T8200), 1 mM phenylmethylsulphonyl fluoride (Solarbio, P8340, Beijing, China), 10 mM dithiothreitol). Extractions were clarified by centrifugation at 12,000 rpm for 30 min at 4 °C, and the protein concentration of the supernatant was adjusted to 1.5 mg/mL. For each precipitation, 500 μg of protein extract was incubated with 200 uL anti-HA magnetic bead suspension (Clontech, Mountain View, CA, USA, Cat. No.635696) for 4 h at 4 °C in a top-to-end rotator. After incubation, the beads were washed four times with ice-cold washing buffer (Clontech, Mountain View, CA, USA, Cat. No.635696) and then eluted by boiling in 2 × SDS loading buffer. Samples were separated by SDS-PAGE and analyzed by immunoblotting with anti-FLAG or anti-HA antibodies.

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.