Ab46540

ChIP assays were performed as previously described (28 (link)) using antibodies specific for RNA polymerase I subunits, RPA116 and RPA194 (generous gift from Ingrid Grummt) as well as a IgG control antibody (abcam ab46540). Briefly, cells were washed and cross-linked for 10 min at 37°C by addition of formaldehyde at a final concentration of 1%. Cells were washed and sonicated with a Branson Sonifier 450 at 20% amplitude with 10-s pulses at 3 min cycle. The chromatin extracts were diluted 10-fold in ChIP dilution and precleared by incubating with 40 μl salmon sperm DNA/protein A-agarose 50% gel slurry for 2 h at 4°C. Proteins were immunoprecipitated at 4°C overnight and washed extensively. DNA-protein cross-links were reversed by incubation at 65°C overnight followed by proteinase K treatment. DNA was recovered by purification with the Qiaquik PCR purification column (QIAGEN).

ChIP assays were performed with 11-day-old estradiol-induced seedlings as previously described (Husbands et al. 2015 (link)), using IgG (abcam ab46540) or anti-GFP (abcam ab290) antibodies. ChIP and input DNA samples were assayed by qPCR using iQ SYBR Green Supermix (Bio-Rad). ZPR3 and ZPR4 regulatory regions assayed in ChIP were selected based on the presence of HD-ZIPIII binding sites predicted by FIMO (https://meme-suite.org/meme/tools/fimo). All experiments were performed at least three independent times. PCR was performed in duplicate, and enrichments calculated relative to input. Student's t-test was used to calculate statistical significance.
Total RNA was extracted from seedlings or infiltrated N. benthamiana leaves using Trizol reagent (Gibco BRL). One microgram of RNA was primed with oligo (dT) and reverse transcribed using the SuperScript III first-strand synthesis kit (Invitrogen). Relative quantification values were calculated based on at least three biological replicates, with ΔCt of ACT2 or B-tubulin serving as normalization controls in Arabidopsis or N. benthamiana, respectively. Wild-type or uninduced values were set to one and PHB variant values either plotted directly or after further normalization to PHB variant levels. Student's t-test was used to calculate statistical significance. ChIP and RT-qPCR primer sequences are listed in Supplemental Data Set 9.

ChIP was performed according to the Affymetrix protocol as previously described.^{39 (link)} Briefly, PR9 cells were incubated with 100 μM ZnSO₄ for 4 h; 5 × 10⁷ cells were then harvested for ChIP assays. A total of 5 × 10⁷ U937 and NB4 cells were harvested for ChIP assays. Cells were crosslinked with 1% formaldehyde and lysed. Cross-linked chromatin was sonicated to fragments with an average size of 200–1000 bp and immunoprecipitated with specific antibodies against RARα (C-20x, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PML (H-238x, Santa Cruz Biotechnology) and rabbit IgG (ab46540, Abcam, Cambridge, UK). The total input as well as immunoprecipitated DNA was analyzed by PCR using following primers: positive primer (region 1) (forward: 5′- TAGTGGAACCTCGGATTGGGT-3′ reverse: 5′- TGATTGGTCAGAGTGTGGCAA-3′), negative primer (region 2) (forward: 5′- TGGTCACAAGTCTCTTTTGCATG-3′ reverse: 5′- CACCTACTTAGGCCTCGAGACAG-3′). Each experiment was performed in triplicate and equivalent results were obtained.

For the preparation of crosslinked chromatin, fresh samples were incubated in PBS with 1% formaldehyde for 10 min at RT. Crosslinking was stopped by the addition of glycine to 125 mM, and cells were washed in PBS. All subsequent procedures were performed on ice, with buffers containing the protease inhibitors (Complete EDTA-free) as previously described (58 (link)). Sonication yielded chromatin fragments of 150–500 bp in length. Immunoprecipitations were performed using antibodies against H4K16ac (Active Motif, 39167), total histone H3 (Abcam, ab1791) as positive control, or IgG antiserum (Abcam, ab46540) as negative control.
After testing the PCR amplification sensitivity of primers, quantitative real-time PCR analysis was performed for each specific region using SYBR^® Green of the StepOnePlus™ Real-Time PCR System (Applied Biosystems). All measurements were performed in triplicate, non-template controls were included, and a calibration curve was determined for each primer set. Three independent experiments were tested for each sequence. Oligonucleotide sequences are listed in Supplementary Table S1.