Fig. 3

IRS1 ubiquitination and degradation depend on neddylation of CUL7. a IRS1 ubiquitination is enhanced in the presence of CUL7. HEK293T cells were co-transfected with GFP-P2A-CBD-His-IRS1, HA-Ubiquitin vectors together with one of the cullin`s family members or empty Myc-vector for control. CBD-His-IRS1 was pulled down with chitin resin for IRS1 enrichment. Quantitation of the levels of pulled-down ubiquitinated IRS1 (b, n = 4 independent experiments) and of total cell homogenates probed with anti-His antibody, and IRS1 expression was normalized to GFP in c (n = 4); ****P < 0.0001, *P < 0.05 versus control, by two-way ANOVA followed by Bonferroni’s multiple comparison test. d Schematic representation of the expression plasmid and the treatment. e Representative immunoblots probed with anti-Ubiquitin, anti-IRS1, anti-Myc, and anti-GFP antibodies. f Depicted is a scattered dot plot showing reduced IRS1 ubiquitination in the presence of MLN-4924 (n = 6). g Schematic of the protocol used to induce IR in DIV11 primary cortical neurons, infected with either AAV9-HA-NEDD8 or AAV9-HA-tag. h, i Synaptic insulin resistance enhanced the association of NEDD8 with CUL7 as revealed with co-immunoprecipitation experiments from primary neurons infected with HA-NEDD8. i Scattered dot plots show quantification of CUL7 in co-IPs normalized to the input (n = 4). j Representative confocal images of dendrites of primary hippocampal neurons infected with AAV9-shRNA CUL7 and AAV9-scr shRNA and immunolabeled with antibodies against pAKT S473 and the synaptic marker Shank3. Original pixel intensities from 0 to 255 are presented as a gradient lookup table. Scale bar, 5 μm. k n = 2566 for group 1; n = 1171 for group 2; n = 1319 for group 3; n = 1194 for group 4; n = 857 for group 5; n = 1059 for group 6. Violin plot representing means of the pAKT at synaptic sites. Numbers refer to the number of spines detected by the Shank 3 mask. ***P < 0.001, **P < 0.01, *P < 0.05 versus control, by two-way ANOVA followed by Bonferroni’s post-hoc test. A two-tailed Student’s t-test was used in (f). n.s. = non-significant. Data are presented as the mean ± SEM