Flag m2 antibody

Antibodies against PTPH1 used in Western blots (WBs) were from Santa Cruz, (Sc-9789), Abgent (AP8426a) or was a monoclonal antibody (2-117) kindly provided by N. Tonks [50 (link)]. The antibodies against the EGFR (D38B1), the pY1068-EGFR (D7A5) and pY1173-EGFR (53A5), pp38MAPK (Thr182/Tyr182, #9211), Myc (71D10), as well as the rabbit IgG isotype control (DA1E) were from Cell Signaling. The anti-Pan-Ras RAS10 (MABS195) antibody was from MerckMillipore. The antibody against Eps15 (no. 610806) was from BD Bioscience. Magnetic M2-FLAG-affinity gel and the FLAG-M2 antibody were from Sigma.

Detached leaves from 4-week-old Col-0 and CCA1OX (35S:CCA1-Flag) plants (Li et al., 2011 (link)) were collected at ZT1. Flag M2 antibody (Sigma) was used for immunoprecipitation. ChIP-qPCR results were first normalized with input sample as follows: cycle threshold (Ct) = Ct_ChIP-Ct_Input. Relative enrichment was then calculated as the ratio of normalized results from 35S:CCA1-Flag relative to the WT control. Primers are listed in Supplementary Table 2.

Protein extraction and immunoblotting was carried out as previously described^{69 (link)}. Briefly, exponentially growing cells in YPD were collected before and after addition of HU at a final concentration of 200 mM. Cells were then fixed with trichloroacetic acid (10% final) for 15 min and whole-cells extracts prepared. Proteins were separated on a SDS–PAGE gel and immunoblots were performed using the FLAG-M2 antibody (Sigma, #F3165) diluted 1:5000 or PGK1 antibody (Novex, #4592250) diluted 1:1,000,000. An uncropped scan of the membrane is included in Supplementary Fig. 5.

Agrobacteria carrying constructs encoding FLAG- and GFP-tagged proteins of interest were co-infiltrated into N. benthamiana leaves. Leaf samples were collected 2 days after the infiltration. One gram of each sample was ground in liquid nitrogen and homogenized in 2.5 mL IP buffer (10-mM HEPES Ph 7.5, 100-mM NaCl, 1-mM EDTA, 10% [v/v] glycerol, and 0.5% [v/v] Nonidet P-40) with 1-mM PMSF, 20-�M MG132, and proteinase inhibitor cocktail for 20 min at 4�C with rotation. Five micrograms of FLAG M2 antibody (F1804; Sigma-Aldrich) was prebound to 60-�L protein G Dynabeads (10003D; Thermo Fisher Scientific) for 30 min in 1� phosphate buffer saline (PBS) buffer with 0.02% (v/v) Tween-20 at room temperature. The beads were washed once with the same PBS buffer and resuspended in Co-IP buffer. After protein extraction, 20 �L of anti-FLAG prebound Dynabeads was added to each sample for another 1.5 h incubation at 4�C with rotation. Dynabeads were precipitated using DynaMagnetic rack (12321D; Thermo Fisher Scientific) and washed twice with Co-IP buffer with 0.5% (v/v) Nonidet P-40 and twice with Co-IP buffer without Nonidet P-40. The IP product was resuspended in 2� SDS sample buffer and used for immunoblotting with rabbit anti-GFP (A11122; Invitrogen) and rabbit anti-FLAG antibody (F7425; Sigma-Aldrich).

For the ChIP-seq in U2OS cells expressing Flag-H2AZ.1 and Flag-H2AZ.2, 100 μg of chromatin supplemented with 10 ng of spike in chromatin (active motif) were used per ChIP experiment. For each reaction, 4 ug of Flag M2 antibody (sigma) and 1 ug of spike in antibody (active motif), were used. About 10 ng of immunoprecipated DNA (quantified with quantiFluor dsDNA system, Promega) were obtained at each time, and samples were submitted to EMBL-GeneCore Heidelberg for sequencing, that was performed by Illumina’s NextSeq 500 technology.
ChIP-seq in K562 cells expressing Flag-H2A.Z.1 and Flag-H2A.Z.2 were performed and analysed as previously described (Jacquet et al., 2016 (link); Lalonde et al., 2013 (link)).