Fig 2: S80 phosphorylation is not required for NF-?B activation. (A) Schematic of the CRISPR/Cas9 targeting strategy used to edit the NFKB1 target locus, highlighting the position of S80 (red); gRNA (yellow); PAM site (blue) and primers used for screening (green arrows). The ssODN template including homology arms (dotted line), HindIII restriction cut site (green triangles) and synonymous codon changes (bold) are shown. Also shown is DNA sequence chromatogram of the NFKB1 target region confirming the S80A point mutation (black box). (B) Whole cell lysates extracted from WT and NFKB1S80A HEK293Ts were analysed by western blot (WB) with the indicated antibodies. (C) WT and NFKB1S80A HEK293Ts were stimulated with 10 ng/ml TNFa for the indicated times prior to lysis. Whole cell extracts were analysed by western blot to detect levels of phosphorylated and total I?Ba protein. (D) WT and NFKB1S80A HEK293Ts were stimulated with 10 ng/ml TNFa for the indicated times prior to lysis. Nuclear and cytoplasmic extracts were analysed by western blot using antibodies against p65 and p105/p50. (E) WT or NFKB1S80A HEK293Ts were left untreated or were stimulated with 10 ng/ml TNFa for 30 min prior to lysis. p50 was immunoprecipitated (IP) from whole cell lysates with anti-p105/p50 antibody and analysed by western blot (WB) with anti-p105/p50 and anti-p65 antibodies as indicated.

Fig 3: Identification of amino acids important for USP7 interaction with p65. A, the 18 amino acids of USP7-derived peptides of interest were sequentially substituted with alanine, probed with GST-p65, and detected by immunoblotting with anti-GST antibody. The peptides are labeled according to Fig. 5. p65 binding was quantified and calculated as a percentage binding of the parent unsubstituted peptide (-) on the same array. Alanine substitutions that resulted in less than 60% binding are indicated by asterisks. The corresponding USP7 amino acid number for first residue of each peptide is shown above each individual substitution peptide series. B, the structure of each Ubl domain with amino acids identified by alanine-scanning experiments indicated in red. The images were generated using Protein Data Bank structure 2YLM. C, HEK293T cells were transfected with p65 and FLAG-tagged USP7, USP7 mutated at residues 757–760, and USP7 lacking the Ubl2 domain (USP7?Ubl2). p65 was immunoprecipitated (IP) from whole cell lysates using anti-p65 antibody and analyzed by Western blotting (WB) with anti-FLAG antibody. The expression of p65 and USP7 in lysates used for immunoprecipitation (input) was measured by Western blotting using anti-FLAG and anti-p65 antibodies. The positions of molecular mass markers are indicated to the right of each Western blot.

Fig 4: Interaction of SIRT6 with RelA/p65.HEK293 cells were transiently transfected with Flag-tagged SIRT6. Cell extracts were prepared and immunoprecipitated with an antibody to RelA/p65 and analysed by Western blotting (WB) with antibodies to RelA/p65 (lanes 1–4) SIRT6 (lane 5–8) and Flag (lane 9–12). Lanes 1,5 and 9 show the anti-RelA/p65 immunoprecipitate (p65 IP), lanes 2,6 and 10 show the non-immunoprecipitated or “flow through” material (FT), lanes 3,7 and 11 show a control immunoprecipitation using only Protein G agarose (p65 control) and lanes 4,8 and 12 are a sample of the total cell extract (input).

Fig 5: The Ubl2 domain of USP7 is required for interaction with p65. A, schematic representation of USP7 mutations used to test interaction with p65. USP7 contains an N-terminal MATH domain, a central catalytic domain (CD), and five C-terminal Ubl domains. B–F, HEK293T cells were transfected with p65 and the indicated FLAG-tagged USP7 plasmids. USP7 was immunoprecipitated (IP) from whole cell lysates using anti-FLAG antibody and analyzed by Western blotting (WB) with anti-p65 antibody. The expression of p65 and USP7 in lysates used for immunoprecipitation (input) was measured by Western blotting using anti-FLAG and anti-p65 antibodies. Western blotting of inputs with anti–β-actin was used to verify equal protein loading. The positions of molecular mass markers are indicated to the right of each Western blot. The data are representative of at least three independent experiments.