Fig 2: Inverse expression patterns of Fbxw7 and its substrate c-Jun detected in cortical neurons during glutamate excitotoxicity.a Primary cultures of cortical neurons were exposed to 200 μM glutamate for the indicated time periods or b cultures were exposed for 2 h to 200 μM glutamate in the presence or absence of 10 μM MG132. a, b Cell lysates were harvested and analyzed by IB. c Primary cultures of cortical neurons were infected with lentiviral particles containing shRNA against either Cdk5 or control (Scramble) at DIV3 and subsequently maintained until DIV 11. Cultures were then exposed to 200 μM glutamate for the indicated time periods. Cell lysates were subjected to IB analysis. d After normalization against GAPDH, relative levels of Fbxw7 at the designated time periods were evaluated against time point 0 and are shown in the graph. Each point represents the mean ± SD from five independent experiments. *p < 0.05; ***p < 0.001. e Primary cultures of cortical neurons were exposed to 200 μM glutamate for up to 6 h. Cell lysates were harvested and analyzed by IB. f After normalization against GAPDH, relative fold intensity of Fbxw7 and c-Jun signal was quantified over the control at time 0 (value = 1). Each point represents the mean ± SD from three independent experiments. **p < 0.01; ***p < 0.001

Fig 3: TFP5-mediated CDK5 suppression reduces HG-induced oxidative stress, inflammatory cytokines, and apoptosis in podocytes. (A) The effect of TFP5 on the level of H2O2 under 24 h HG treatment. H2O2 was measured by hydrogen peroxide test kit. (B) The detection of ROS after TFP5 treatment. ROS Level was assessed using the probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) following TFP5 treatment. (C–E) The effect of TFP5 of the mRNA level of (A) H2O2, (B) ROS, (C) Caspase-3, (D) Bax, and (E) Bcl-2 expression of MPC-5 after 24 h HG treatment. (F) The effect of TFP5 on the protein level of CDK5, p35/25 and apoptosis-related cleaved caspase-3 in MPC-5 after 24 h HG treatment. (G) The effect of TFP5 on HG-induced apoptosis of MPC-5 cells. PI was used to separate the viable cells and Annexin-FITC was used to identify the apoptotic cells. *p < 0.05, **p < 0.01, ***p < 0.001. A p value less than 0.05 was considered as statistically significant.

Fig 4: Both Ser349 and Ser372 are the major sites of Fbxw7 that are phosphorylated by Cdk5/p25.a Scheme for putative Cdk5-phosphorylaton sites of Fbxw7 (S/TPXK/R). b In vitro kinase assay to detect the direct phosphorylation sites of Fbxw7 by Cdk5/p25. Purified GST-Fbxw7 wild-type (WT) protein (1 µg) or one of the GST-Fbxw7 site-specific mutant proteins were incubated with 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. c HEK293 cells were transfected with Flag-Fbxw7 WT or one of the Flag-Fbxw7 site-specific mutant proteins in the presence or absence of HA-Cdk5/Myc-p25. For comparison with the p-Fbxw7 signal, HEK293 cells were transfected with Flag-Fbxw7 2A mutant plus HA-Cdk5/Myc-p25. Cell lysates were incubated with Flag-conjugated beads for IP. Immunoprecipitates and WCL were resolved by SDS-PAGE analyzed by IB analysis. Phospho-Fbxw7 signal was visualized with an anti-phospho-Cdk5-substrate antibody

Fig 5: Cdk5 negatively regulates Fbxw7 protein expression level.a HEK293 cells were transfected with Flag-Fbxw7 alone or in combination with either HA-Cdk5 WT/Myc-p25 or HA-Cdk5 DN/Myc-p25. IB analysis was conducted using the indicated antibodies. b Relative intensity of Flag-Fbxw7 was calculated over the control (only transfected with Fbxw7; value = 1) after normalization against GAPDH. Bar represents the mean ± SD of three independent experiments. *p < 0.05; ***p < 0.001; n.s not significant. c HEK293 cells were exposed to 10 μM roscovitine for 48 h. IB analysis was conducted with the indicated antibodies. d Relative intensity of Flag-Fbxw7 intensity was calculated over the control (value = 1) after normalization against GAPDH. Bar represents the mean ± SD of three independent experiments. *p < 0.05; ***p < 0.001; n.s not significant. e HEK293 cells transfected with Flag-Fbxw7 alone or in combination with HA-Cdk5/Myc-p25 were treated with or without 10 μM MG132 for 8 h. Cell lysates were analyzed by IB using the indicated antibodies. f Relative intensity of Flag-Fbxw7 was calculated over the control (value = 1) after normalization against GAPDH. Bar represents the mean ± SD of three independent experiments. **p < 0.01; ***p < 0.001; n.s not significant