Cycloheximide ch

Six-month-old plants of sweet orange “Pera” were obtained from certified nurseries and kept in a growth room at 25–28°C with a 14 h light photoperiod. For plant infiltration, Xc strains were inoculated from single colony plates and grown over night at 28°C in liquid LB without NaCl. Cells colonies were suspended in sterile water to an optical density at 600 nm (OD₆₀₀) of 0.1 (nearly 10⁶ CFU/ml). Leaves were infiltrated with bacterial suspensions in water or, in 50 μM cycloheximide (Ch) (Sigma-Aldrich, USA). Water and Ch only were independently infiltrated as mock controls. For quantitative PCR (qPCR) assays, fully expanded “Pera” leaves were infiltrated with a water suspension (OD₆₀₀ = 0.1) of wild type Xc strain 306, or its pthA4-deletion mutant derivative
[23 (link)].

The following antibodies were from Cell Signaling Technology: anti-Casp8 (9746), anti-Cap3 (9664), anti-cIAP2 (3136), anti-pSAPK/JNK (JNK1/2, 4668), anti-pIκBα (9246), anti-Caveolin1 (3267), and anti-cFLIP (56343). The following antibodies were from Abcam: anti-pMLKL (187091), anti-RIPK3 (72106), anti-pRIPK3 (209384), and anti-FADD (108601). Anti-Ub (S.C8017), and protein A/G-conjugated beads were from Santa Cruz Biotechnology, anti-total MLKL (M6697) was from Sigma-Aldrich, anti-RIPK1 (610459) was from BD Medical Technology, and anti-cIAP1 was a gift from of John Silke (Walter and Eliza Hall Institute of Medical Research). z-VAD-fmk was from Enzo Life Sciences, RIPK1 inhibitor GSK’963, RIPK3 inhibitor GSK’840 and IAP antagonist SMAC007, as well as the pRIP1 S166-specific antibody, were provided by GlaxoSmithKline^{34 (link)}. IAP antagonist BV6 was provided by Domogoj Vucic (Genentech), recombinant human TNF was from R&D or from PeproTech, Flag-Tagged TNF was from Enzo, necrosulfonamide was from CalBiochem, TLCK (Tosyl-L-lysyl-chloromethane hydrochloride) was from Abcam, cycloheximide (CH) was from Sigma-Aldrich, and Carfilzomib was from BioVision.