Anti gfp g6539

TNF-α and z-VAD-fmk were purchased from R&D. Cyclohexamide (CHX) from Sigma. Smac mimetic was a gift from S. Wang (University of Michigan, Ann Arbor, Michigan, USA). TRAIL from Novoprotein. FasL from Upstate Biotech. CH11 from MBL Medical and Biol Lab.
All antibodies are at concentration of 1 μg ml⁻¹ and used at 1:1,000 dilution unless otherwise stated. Anti-RARγ (C-15) (sc-550) for human, anti-RARα (C-20) (sc-551), anti-caspase-8 (C-20) (sc-6136), anti-cIAP2 (sc7944) and anti-Fas (C-20) (sc-715) from Santa Cruz; anti-RIP1 (610459) and anti-FADD (610400) from BD Biosciences; anti-RARγ1 (ab5905) for mouse, anti-RIP3 (ab72106), anti-MLKL (ab184718) for human, anti-MLKL (ab172868) for mouse and anti-cIAP1 (ab2399) from Abcam; anti-RIP3 (2283) for mouse from ProSci, anti-TRADD (05-473) from Upstate; anti-TRAF2 (MAB3277) and anti-TNFR1 (AF-425-PB) from R&D; anti-Actin (A3853) (dilution 1:10,000), anti-FLAG (F9291) (dilution 1:5,000) and anti-GFP (G6539) (dilution 1:5 000) from Sigma; anti-V5 (R960-25) (dilution 1:5,000) from Invitrogen; anti-PARP1 (BML-SA250-0050) from Enzo Life Science; anti-GAPDH (NB300-22) (dilution 1:5,000) from Novus Biologicals; anti-cleaved caspase-8 (9496), anti-CYLD (4495), anti-RIP1 (137451) and anti-p-RIP1(65746) from Cell Signaling Technology; anti-DsRed (632392) (dilution 1:5,000) from Clontech.

For each ChIP library, 50 ml of Mabs-S-Δlsr2-Clsr2-FLAG was grown until an OD₆₀₀ of 0.5 and fixed in 1% formaldehyde (Euromedex) and lysed using a Precellys grinder (3 cycles: 6,700 rpm −3 × 20 s ON/60 s OFF, Bertin Technologies) and VK05 beads. The bacterial chromatin was sheared in 100–300 bp fragments using a Bioruptor Pico (Diagenode). Chromatin Immunoprecipitation was performed as described previously (Grainger et al., 2006 (link)) using IgG M2 anti-FLAG (Sigma, F1804) and Anti-GFP (G6539, Sigma) mouse monoclonal antibodies attached to proteins A/G coupled to magnetic beads (Thermo fisher). The immunoprecipitated DNA and 1% of the total input were reverse-crosslinked and eluted using the iPure v2 kit (Diagenode). The quantitative PCR tests were performed using the SsoFast evergreen supermix (Bio-Rad). Enrichment of Lsr2 binding at the GPL operon operator was calculated using the “Percent Input” method using the primers MAB_4100c_qPCR1 and MAB_4100c_qPCR3 (Supplementary Table S1).