Fig. 6

Increased S6 phosphorylation and protein synthesis in the patient cell line are decreased with a p110δ-selective inhibitor. a Protein synthesis assays using metabolic labeling of nascent peptide chains with puromycin followed by puromycin-specific Western blots show that basal protein synthesis rates in the cell line from the autistic patient (A4) are increased compared to cell lines from his unaffected sibling (A4-S). The p110δ-selective inhibitor IC87114 (1 nM, 15 min) reduces protein synthesis rates in the autistic patient but does not affect protein synthesis in the unaffected sibling (n = 7, two-way ANOVA F _interaction(1,6) = 10.73, p _interaction = 0.017; F _treatment(1,6) = 9.34, p _treatment = 0.022; F _autism(1,6) = 1.20, p _autism = 0.316; Sidak’s post hoc analyses *p = 0.010, ^# p = 0.011). Example Western blot is shown on the right; “no puro” samples show background staining without puromycin treatment of the cells. b In contrast, the p110β-selective inhibitor TGX-221 (1 μM, 15 min) does not affect protein synthesis rates in cell line A4 (n = 4, paired t test, t(3) = 0.34, p = 0.76). Example Western blot is shown on the right; samples were loaded in duplicates. c Selective inhibition of p110δ with IC87114 decreases S6 phosphorylation in the cell lines from both the autistic individual and his unaffected sister. Quantification of ELISAs is shown on the left (n = 4, two-way ANOVA with Sidak’s post hoc tests F _autism(1,3) = 35.01, p _interaction = 0.001; F _treatment(1,3) = 163.2, p _treatment = 0.001; F _autism(1,3) = 4.15, p _autism = 0.135; *p = 0.003, ^# p = 0.0005; ^$ p = 0.0019). Example Western blot is shown on the right. d Similarly, selective inhibition of p110δ with TGX-221 reduces S6 phosphorylation in cell line A4 as measured by Western blotting (n = 5, paired t test, t(4) = 5.75, p = 0.005). Representative Western blot is shown on the right; samples were loaded in duplicates. Shown are means + SEM