Ab167154

Two clones of A20 knockout (A20^-/-) A549 cells were produced using the Transcription Activator-Like Effector Nucleases (TALENs) technology [33 (link)] and the plasmids Human-H27583_TNFAIP3_TALEN-L and Human-H27583_TNFAIP3_TALEN-R (Talen Library Resource, Seoul National University), as previously described [31 (link)]. In brief, A549 cells (parental cells) were transfected with those plasmids encoding sequence-specific DNA-cleaving nucleases against A20. Three days after transfection, the cells were trypsinized and cloned by limiting dilution in 96-well plates at a density of 1 cell per well. Single-cell clones were selected, cloned a second time by limiting dilution, and expanded to generate stocks. Screening for A20^-/- clones was done by PCR amplification and DNA sequencing using the following primers: forward (5′- CCTTTGCAACATCCTCAGAAG-3′) and reverse (5′- ACTAACCAAGCAAGTCACAGAAC-3′). A20^-/- clones were checked by Western blotting using an A20 specific antibody (Ab167154, Abcam, Cambridge, UK). Two A20^-/- clones (KO-1 and KO-2) were selected. In addition, one wild-type (A20^+/+) clone (WT-1) that underwent the same process of transfection and cloning, but in which the A20 gene was not disrupted, was selected as control (Supplementary Materials, Figure S1).

For immunoprecipitation (IP), whole-cell extracts were lysed in an IP buffer composed of 1% (vol/vol) NP-40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.5) and the complete protease inhibitor cocktail (Roche). Cell lysates were collected and incubated with 1 μg of the corresponding antibodies together with protein G Plus-Agarose Immunoprecipitation reagent (Santa Cruz). After overnight incubation at 4 °C, agaroses were washed five times and boiled with the IP buffer for SDS-PAGE. For immunoblotting analysis, cells were lysed with a lysis buffer (150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 50 mM Tris-HCl, pH 7.5) supplemented with the protease inhibitor cocktail (Roche), and were incubated with the following antibodies: anti-FLAG (Proteintech, 20543-1-AP), anti-HA (Proteintech, 51064-2-AP), anti-His (Proteintech, 66005-1-lg), anti-RIP1 (Santa Cruz, sc-133102), anti-RIP3 (Santa Cruz, sc-374639), anti-RIP1(Santa Cruz, sc-133102), anti-Myc (Proteintech, 67447-1-Ig), anti-pRIP3 (Abcam, ab205421), anti-mouse pMLKL (Abcam, ab196436), anti-human pMLKL (Abcam, ab206336), anti-MLKL (Abcam, ab243142), anti-Actin (Abcam, ab8226), and anti-TRIM25 (Abcam, ab167154).