Increase of GluA1 palmitoylation is associated with synaptic upscaling in Ppt1^−/− neurons. A, Representative immunoblot of APEGS assay-processed cortical neurons lysates probing for GluA1 and β-actin. Representative blot for Ppt1 (input) also shown. Neurons were treated with bicuculline (20 μm), vehicle control (DMSO, final concentration 0.1%), or TTX (1 μm) for 48 h before APEGS assay. B, Representative immunoblot band density plots displaying distinct peaks for the palmitoylated (green arrow) and nonpalmitoylated fractions (orange arrow), which were quantified separately and expressed a ratio of palmitoylated/nonpalmitoylated GluA1. C, Quantification of the ratio of palmitoylated/nonpalmitoylated GluA1 levels, normalized to β-actin loading control. Two-way ANOVA: interaction genotype × treatment (F_(2,24) = 4.211, *p = 0.0271); main effect of genotype (F_(1,24) = 9.559, **p = 0.0050); main effect of treatment (F_(2,24) = 11.80, ***p = 0.0003). Tukey's multiple comparison indicated on graph: **p = 0.0017 Ppt1^−/− Veh versus Ppt1^−/− TTX; **p = 0.0065 WT TTX versus Ppt1^−/− TTX. n = 5 independent cultures. Data represent mean ± SEM. D, Quantification of the Ppt1 levels, normalized to β-actin loading control. Two-way ANOVA: interaction genotype × treatment (F_(2,24) = 13.07, ***p = 0.0001); main effect of genotype (F_(1,24) = 847.4, ****p < 0.0001); main effect of treatment (F_(2,24) = 13.07, ***p = 0.0001). Tukey's multiple comparison indicated on graph: ***p = 0.0004 WT Veh versus WT TTX. n = 5 independent cultures. Data represent mean ± SEM.