Ab177790

After cell culture, cells were washed with phosphate‐buffered saline (PBS; Merck) and fixed with 4% w/v paraformaldehyde (Sigma‐Aldrich) for 5 min at 37 °C. After a washing step, cells were permeabilized with 0.01% v/v Triton X‐100 (Acros Organics) and blocked with goat serum (1:100; Sigma‐Aldrich) in PBT (PBS + 0.02% Triton‐X‐100, 0.5% BSA) for 1 h. Afterward, cells were incubated with the primary antibody in PBT for 1 h. After a washing step, cells were incubated with a secondary antibody conjugated to an Alexa Fluor (1:500; ThermoFisher) in PBT. In conjunction, phalloidin conjugated to an Alexa Fluor (1:500; ThermoFisher) in PBT was added to the sample for 1 h. After washing, the nucleus was counterstained with Hoechst 33258 (1:1000; Sigma‐Aldrich) for 10 min. After a subsequent washing step, surfaces were mounted on glass cover slides with mounting media (Dako). All washing steps were performed in triplicate with PBT. Primary antibodies used in this study are: anti‐acetyl‐histone H3 antibody (1:200; Millipore; 06–599), anti‐acetyl histone H4 antibody (1:200; Abcam; ab177790), anti‐H3K9Me2 (1:200; Abcam; ab1220), anti‐nucleophosmin antibody (1:200; Abcam; ab37659), anti‐fibrillarin (1:100; Abcam; ab4566), and anti‐nucleolin antibody (1:200; ThermoFisher; ZN004).

The following primary antibodies were used for immunoblotting analysis: anti-KAT7 (ab190908, Abcam), anti-PKD1 (90039S, Cell Signaling Technology-CST), anti-HA (CST, 3724S), anti-Flag (Sigma, F7425), anti-pMOTIF PKD1 (4381, CST), anti-phosphoserine (ab9332, Abcam), anti-phosphothreonine (CST, 9381S), anti-Tubulin (BS1482M, BioWorld), anti-phospho-PKD1(ser744/748) (CST, 2054), anti-GAPDH (Bioworld, AP0063), anti-Myc, anti-GST(Santa Cruz, sc-965), anti-mcm2 (ab133325, Abcam), anti-mcm6 (ab201683, Abcam), anti-H4 (13919S, CST), anti-H4acK5, K8, K12, K16 (ab177790, Abcam), anti-H3.

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.