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. 2019 Oct 10;47(21):11151–11163. doi: 10.1093/nar/gkz873

Figure 3.

Figure 3.

S80 phosphorylation is not required for NF-κB 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 TNFα for the indicated times prior to lysis. Whole cell extracts were analysed by western blot to detect levels of phosphorylated and total IκBα protein. (D) WT and NFKB1S80A HEK293Ts were stimulated with 10 ng/ml TNFα 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 TNFα 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.