Ab18181

Whole-cell extracts were prepared in IPH lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 5mM ethylenediaminetetraacetic acid (EDTA), 0.5% NP40). Nuclear extracts were prepared as follows: cells were first lysed in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, protease inhibitors) and centrifuged at 10 000 g for 5 min. Buffer B (20 mM HEPES pH7.9, 0.4 M NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, protease inhibitors) was then added to the nuclear pellet fraction, which was shaken vigorously for 2 h and centrifuged at 10 000 g for 10 min. The supernatant (nuclear fraction) was aliquoted and stored at –80°C until used. All procedures were performed at 4°C. For DNAse treatments, nuclear extracts were incubated with an excess of DNAse I for 1 h at 37°C prior to immunoprecipitation. The effectiveness of DNAse treatment was checked on plasmidic DNA (see Supplementary Figure S1).
Standard procedures were used for immunoprecipitation and western blotting (10 (link)). The primary antibodies used in these experiments were directed against the following: control immunoglobulin G (IgG) (sc-2027; Santa Cruz), HA (ab18181; Abcam), GAL4 (sc- 510; Santa Cruz), DNMT3A (sc-20703; Santa Cruz), PADI4 (ab18181; Abcam), Citrulline (ab100932; Abcam), Citrulline H3 (ab1791, Abcam), HDAC1 (pAb-053–050; Diagenode), Actin (Sigma; A5316) and TBP (ab818, Abcam).

Salmonella cultures were grown in LB to the stationary phase under microaerobic conditions at 37°C or 41°C, as indicated. The cultures were OD₆₀₀ normalized, centrifuged, and pellets were resuspended in 1× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Boiled samples were separated on 10% or 12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories). Blots were probed with anti-HA tag antibody (Abcam; ab18181, diluted 1:1,000); anti-RpoD antibody (Santa Cruz Biotechnology; SC56768, diluted 1:2,000) or anti-DnaK (Abcam; ab69617, diluted 1:10,000), when the marketing of the anti-RpoD antibody was discontinued by the manufacturer. Goat anti-mouse antibody conjugated to horseradish peroxidase (Abcam; ab6721, diluted 1:5,000) was used as a secondary antibody, followed by detection with enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia).