Fig. 1

P2X7R activation in N2a cells increases proteasomal chymotrypsin-like and post-glutamyl-like activities by increasing the proteasomal subunits β5 and β1 translation and transcription and promoting its later assembly to 20S proteasomal core via IP3K/GSK3/Nfr2. A Representative Western blot and quantification of UPS reporter levels in Ub^G76V-YFP transfected N2a cells stimulated with 200µM BzATP or vehicle solution (CTL), in the presence or absence of selective P2X7R antagonist 40µM A438079 (n = 4 in triplicated). B Genetic knockdown of P2X7 avoids BzATP-induced Ub^G76V-YFP accumulation in N2a cells (n ≥ 3 in duplicated). Sample loading was normalized with anti-α-tubulin (α-Tub) antibody. Graphs show the quantification of Ub^G76V-YFP levels. 100% corresponds to the Ub^G76V-YFP protein expression detected in Ub^G76V-YFP transfected cells (A) or cells co-transfected with Ub^G76V-YFP plus shRNA P2X7 (B) treated with vehicle solution. C Percentage of N2a cells survival treated with 10 µM MG132 for 24 h in the presence or absence of 40 µM A438079 (n = 4 in duplicated). Measurement of chymotrypsin-like (D) and postglutamyl (E) peptidase activities assayed in N2a cells stimulated with 200µM BzATP in the presence or absence of 40µM A438079 (n = 3 in triplicated). 100% value corresponds to the activity detected in vehicle-treated N2a cells. In all reactions, the specificity of the fluorogenic reaction was verified by the addition of the MG132 proteasome inhibitor. **p ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001 using a one-way ANOVA followed by Tukey’s post hoc test. F Western blot analysis of β1 and β5 proteasome subunits expression in N2a cells stimulated with 200µM BzATP or vehicle solution. Quantitative PCR analysis of β5 (psmb5, G) and β1 (psmb6, H) mRNA levels in vehicle- (CTL) or BzATP-treated N2a cells. Values were normalized to endogenous GAPDH as a loading control (n ≥ 3 in duplicated). I Representative Western blot image and quantification of β5 and β1 subunits immunoprecipitated with MCP-8017.3 antibody. Protein amount was normalized to 20S core (n ≤ 3 in duplicated). *p ≤ 0.05; **p ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; using an unpaired two tailed Student’s t-test. J Representative Western blot and quantification of UPS reporter levels in Ub^G76V-YFP transfected N2a cells stimulated with 200µM BzATP in the presence or absence of selective inhibitors of intracellular kinases, the PI3K inhibitor 50 µM LY294002 (LY), or the GSK3β kinase 5 µM SB216763 (SB). Sample loading was normalized with anti-α-tubulin antibody. 100% corresponds to Ub^G76V-YFP protein expression detected in cells treated with vehicle solution (CTL). *p ≤ 0.05; *** P ≤ 0.001; using a two-way ANOVA followed by Tukey’s post hoc test. K Representative Western blot image and quantification of Nfr2 transcription factor levels in N2a cells stimulated with 200µM BzATP or vehicle solution (n ≥ 4 in duplicated). *p ≤ 0.05 using an unpaired two-tailed Student’s t-test. L Western blot and quantification of β1 and β5 expression levels in N2a cells stimulated with vehicle solution (CTL) or 200 BzATP in the presence or absence of selective Nfr2 stimulator 10 µM sulforaphane. Sample loading was normalized with anti-α-tubulin antibody. 100% corresponds to β1 and β5 expression detected in cells treated with vehicle solution (CTL). Data in bar graphs represent mean ± s.e.m. *p ≤ 0.05; ** P ≤ 0.01; using a one-way ANOVA followed by Tukey’s post hoc test