Fig. 2

MLN-4924 prevents degradation of IRS1 upon induction of synaptic IR. a Schematic drawing represents the timeline of the experiment and corresponding treatments of primary neurons at DIV21. The proteasome inhibitor MG-132 (20 µM) or MLN-4924 (1 µM) was applied for 24 h. b, f Membranes were probed with anti-IRS1 or anti-InsR antibodies. All blots were probed with β-actin antibody to control for loading. Scatter dot plot represents analysis of IRS1 (n = 7, c, cortical culture preparations) and InsR (n = 7, d) levels upon treatment with MG-132, as well as IRS1 (n = 6, g) and InsR (n = 6, h) levels upon treatment with MLN-4924. i The scheme illustrates the protocol and corresponding treatments. j Representative immunoblots of cortical neuron homogenates probed with anti-pAKT S473 and anti-pan-AKT as well as anti-pInsR and anti-pan-InsR antibodies. β-Actin was used as a loading control. pAKT/AKT ratios (n = 8, l) and pInsR/InsR ratios (n = 8, k) decreased in response to 15-min stimulation with insulin in conditions of synaptic IR, which was significantly rescued in the presence of MLN-4924. Statistical analysis refers to the groups that have received the acute insulin stimulation only. m Representative confocal images of dendrites of primary hippocampal neurons immunolabeled with antibodies against pAKT S473 and synaptic marker Shank3. Original pixel intensities from 0 to 255 are presented as a gradient lookup table. Scale bar, 10 µm. n Scatter dot plot representing the mean immunoreactivity of synaptic pAKT. Data were obtained from at least two independent coverslips and at least two independent cell cultures. n = 23 for group 1; n = 25 for groups 2, 3 and 4. The numbers indicate the number of dendrites acquired from different neurons. ***P < 0.001, **P < 0.01, *P < 0.05 versus control, by two-way ANOVA followed by Bonferroni’s post-hoc test. n.s. = non-significant. Data are presented as the mean ± SEM