Fig. 1: Cdk5/p25 phosphorylates Fbxw7.

a HEK293 cells were transfected with Flag-Fbxw7 in combination with either HA-Cdk5/Myc-p25, HA-Cdk5/Myc-p35, or neither for the control. Cell lysates were incubated with Flag-conjugated beads for immunoprecipitation (IP). Immunoprecipitates and whole cell lysates (WCL) were resolved by SDS-PAGE and followed by immunoblot analysis (IB). Phospho-Fbxw7 signal was visualized with an anti-phospho-Cdk5-substrate antibody. GAPDH was used as a loading control. b HEK293 cells were transfected with Flag-Fbxw7 in combination with either HA-Cdk5 WT/Myc-p25 or HA-Cdk5 dominant-negative D144N (DN)/Myc-p25. WCL were used to IP using Flag-conjugated beads and IB analysis was done using the indicated antibodies. c HEK293 cells were exposed to 10 μM roscovitine for 48 h. Immunoprecipitates were purified with an anti-Flag antibody and IB was conducted with the indicated antibodies. d After normalization to Flag-Fbxw7, the relative fold intensity of the phospho-Fbxw7 signal by Cdk5/p25 were measured in samples treated with or without roscovitine (value = 1). Bar represents the mean ± SD of three independent experiments. ***p < 0.001. e Purified GST-Fbxw7 protein (1 µg) was incubated with or without 0.1 µg recombinant His-Cdk5/GST-p25 protein in the presence of [γ-32P] ATP. Reaction mixtures were resolved by SDS-PAGE and subjected to autoradiography. f After normalization to GST-Fbxw7, the relative fold intensity of the phospho-Fbxw7 signal by Cdk5/p25 was measured in samples treated with or without roscovitine (value = 1). The bar represents the mean ± SD of three independent experiments. ^***p < 0.001