Anti h3k9me2

Chromatin immunoprecipitaion assays for reporter gene promoters and a HDA6 endogenous target (At5g41660) were performed with 2-week-old seedlings. Around 1 g of fresh seedlings was harvested and cross-linked with 1% formaldehyde under vacuum for 15 minutes, and then quenched with a 2 M glycine solution and vacuumed for additional 5 minutes. After removing the formaldehyde, the sample was washed with sterilized deionized water three times. Further grinding, immunoprecipitation as well as reverse cross-linking were performed using an EpiQuick plant ChiP kit (Epigentek Inc, Farmingdale, NY) following the manual provided by the manufacturer. Anti-H3K9me2 was from Epigentek, and antibodies against H3K9ac and H3K27ac were from Millipore. The purified genomic DNA fragments were used as templates for qPCR46 47 (link)48 (link)49 50 (link)51 (link).

Antibodies were purchased from the following suppliers: anti-H3K9me2, anti-H3K27me3 and anti-H3K9ac (EpiGentek, Farmingdale, NY); anti-H3K4me3, anti-H3K4me1 and anti-β-actin (Abcam, Cambridge, UK); negative rabbit IgG (Cell Signaling Technologies); anti-PRDM1 (sc-66015, Santa Cruz Biotechnologies, Dallas, TX); anti-TP53 (P6874 from Sigma and sc-98 from Santa Cruz); anti-mouse HRP-conjugated and anti-tubulin (Sigma); and anti-FOXA1 from Santa Cruz. Recombinant human TP53 protein (ref. 506165) was obtained from Calbiochem (San Diego, CA). The inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) was purchased from Sigma-Aldrich and dissolved in DMSO.

To determine the ratio of H3 K9me2/H3 total N2 (WT) and CL2006 animals were incubated in liquid culture with vehicle (DMSO 1 %), or 0.1 μM of G9a inhibitor in a 96‐well plate format as already mentioned. For chronic treatment, four‐day‐old animals were collected with M9 buffer. Histone extraction was performed following the manufacturer's instructions (EpiQuik Total Histone Extraction HT Kit, EpiGentek, #OP‐0007‐192). The samples were resolved in a 14 % SDS‐gel, as previously described.^[12] To capture chemiluminescence signals were used Amersham Imager 680 and Western blot quantifications were performed using ImageLab software (Bio‐Rad). Immunoblots were probed with anti‐H3 K9me2 (1 : 1000) (Epigentek, #A‐4035), and anti‐H3 total (1 : 1000) (Cell signaling, #9715).