Ab9045

Immunostaining of slides was done by an InsituPro VSi staining system (Intavis, Cologne, Germany) as previously described, with minor modifications (Borg et al., 2019 (link)). Slides were washed for 10 min with TBS with 0.1% Tween-20 (TBST) five times then blocked with blocking buffer (1× TBS, 0.1% Tween-20, 2% bovine serum albumin (BSA), 5% normal goat serum) twice for 30 min each. One antibody per slide (H3K27me3 Millipore #07-449 RRID:AB_310624; H3K36me3 Abcam ab9050 RRID:AB_306966; H3K9me1 Abcam ab9045 RRID:AB_306963) was diluted 1:100 and slides were incubated for 6 hr. After washing with TBST six times for 10 min each, slides were incubated with a 1:500 dilution of secondary antibody (Goat Anti-Rabbit IgG H&L, Alexa Fluor 488, ab150077 RRID:AB_2630356; Goat Anti-Mouse IgG H&L, Alexa Fluor 568, ab175473 RRID:AB_2895153; Goat Anti-Rabbit IgG H&L, Alexa Fluor 647, preadsorbed, ab150083 RRID:AB_2714032) and slides incubated for 2 hr. After eight 10 min washes with TBST, slides were dried and counterstained with 1.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) and mounted in Vectashield antifade mounting medium with DAPI (Vector Laboratories, Piedmont, Italy) before being sealed with a coverslip and nail varnish.

Prior to labeling, 2 × 10⁶ pYD1-mSA-containing yeast cells were washed and pelleted. The samples were then incubated with either an H3K9me1, H3K9me2, or H3K9me3 antibody for 30 min with rotation at 4 °C in 100 µL of 0.1% PBSA (ab1220 1:200, ab9045 1:200, ab8898 1:250, Abcam; Cambridge, UK). The samples were then washed with 0.1% PBSA and incubated with a secondary antibody for 10 min with rotation at 4 °C in the dark in 100 µL 0.1% PBSA (ab150075 1:250, ab150107 1:250, Abcam; Cambridge, UK). The samples were washed in 0.1% PBSA and run on a MACSQuant VYB cytometer using a 561 nm laser and a 661/20 nm filter. Flow cytometry data were analyzed with the FlowJo software.

Western blotting was performed using the methods reported by Legartová et al. (2014) and Franek et al. (2016) [43 (link),6 (link)]. We used the following primary antibodies: anti-53BP1 (#ab21083, Abcam), anti-α-tubulin (#ab80779, Abcam), anti-MDC1 (#ab11169, Abcam), anti-MDC1 (#ab41951, Abcam), anti-phosphorylated histone H2AX (γH2AX; phospho S139; #ab2893, Abcam), anti-HP1β (#MAB3448, Merck), anti-H3 (#ab7091, Abcam), anti-H3K9me1 (#ab9045, Abcam), anti-H3K9me2 (#ab1220, Abcam), anti-H3K9me3 (#ab8898, Abcam), anti-histone H3K9ac (#06-942, Merck), anti-histone H4K20ac (#701778, ThermoFisher Scientific), anti-histone H4K20me2 (#A-4047-050, Epigentek, Lab Mark a.s.), anti-histone H4K20me3 (#A-4048-050, Epigentek, Lab Mark a.s.). As secondary antibodies, we used anti-rabbit IgG (#A-4914, Merck, Germany; dilution 1:2000), anti-mouse IgG (#A-9044, Merck; dilution 1:2000) and anti-mouse IgG1 (#sc-2060, Santa Cruz Biotechnology, Dallas, Texas, USA; dilution 1:1000).

For Western blot analysis, histone-enriched fractions were extracted from wild type (WT), mutant, and transgenic leaves as described previously (Huang et al., 2007 (link)). Antibodies used in Western blot are: anti-H3K27me3 (07-449, Millipore), anti-H3K27me2 (ab24684, Abcam), anti-H3K27me1 (ab113671, Abcam), anti-H3 (ab1791, Abcam), anti-H3K4me3 (07-473, Millipore), anti-H3K4me2 (07-430, Millipore), anti-H3K4me1 (07-436, Millipore), anti-H3K9me3 (ab8898, Abcam), anti-H3K9me2 (07-441, Millipore), anti-H3K9me1 (ab9045, Abcam) H3K36me1 (ab9048, Abcam), anti-H3K36me2 (ab9049, Abcam), and anti-H3K36me3 (ab9050, Abcam). Anti-SDG711 was prepared by immunizing rabbits with SDG711 protein produced in Escherichia coli (in pET-28a vector) and purified with His-tag protein purification beads (V8550, GE Healthcare). The anti-serum was affinity-purified with protein-A agarose beads purchased from Millipore (16-157).