A49 binding to β-TrCP WD40 domain requires its N-terminal region.

<p>(A) Domains of β-TrCP and truncations generated. β-TrCP.ΔWD40 (1–250), β-TrCP.ΔF box (251–569). (B) HeLa cells were cotransfected with nTAP-A49 together with HA-tagged WT β-TrCP, ΔWD40 β-TrCP, ΔF-box β-TrCP or SINTBAD. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. WCL (2.5%) of each sample was analysed. (C) HeLa cells were cotransfected with myc-tagged β-TrCP together with nTAP-tagged WT A49, S7/12A A49, 7/11/12A A49, S7/12E A49 or C6 as a negative control. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. A fraction (2.5%) of each whole cell lysate (WCL) was analysed. (D) HEK293T cells were transfected with an NF-κB luciferase reporter, a renilla luciferase reporter and 100 ng of either nTAP-tagged WT, S7/12A or S7/12E A49 plasmids, or empty vector (EV). After 24 h cells were stimulated for 6 h with 20 ng/ml TNFα and luciferase activity was measured. Data are presented as mean ± SD and show one representative experiment of at least three, each performed in triplicate. *p<0.05, **p<0.01 or ***p<0.001 comparing conditions pair-wise as indicated. (E) Lysates from TNFα-stimulated samples from the reporter gene assays were fractionated and immunoblotted for FLAG and tubulin. WCL (10%) of each sample was analysed. Data shown in (B–C) are one representative experiment of at least three showing indistinguishable results.