Figure 4.

NLRP1 and Casp1 activate Casp6 in serum-deprived and BzATP-treated neurons. Casp6 VEIDase activity (a and d), TubΔCasp6, full-length tubulin (FL Tub), β-actin western blots (b and e) and IL-1β production (c and f) in serum-deprived (a,b, and c) or BzATP-treated (d,e, and f) neurons in the absence (DMSO control) or presence of 5 μM Casp1 Z-YVAD-fmk. Data represent the mean and S.E.M. of three (a and c) and four (d and f) independent neuron cultures. One-way ANOVA (P=0.002 in a, P=0.004 in c, and P=0.02 in d and f) followed by a Tukey–Kramer post hoc analysis (*P<0.05 and **P<0.01) relative to untreated DMSO control. (g) Casp6 VEIDase activity in neurons treated with 1 μM scrambled siRNA or siRNAs against Casp1 or NLRP1 followed by normal culture conditions (control), serum deprivation or 500 μM BzATP for 1 h. Data represent the mean and S.E.M. of four independent experiments. Scrambled siRNA-treated control was arbitrarily placed at one and other values within each experiment expressed relative to the scrambled siRNA control levels. One-way ANOVA (P=0.0106) followed by a post hoc Tukey–Kramer confirms increased Casp6 activity in serum-deprived neurons (*P≤0.05) and return to normal in siNLRP1 and siCasp1 serum-deprived neurons. Independent unpaired t-test shows ^#P<0.05 between scrambled siRNA and siNLRP1 or siCasp1 in serum-deprived conditions, and between scrambled siRNA and siNLRP1 in BzATP-treated neurons. (h) RT-PCR of NLRP1 and CASP1 mRNA in siNLRP1 and siCASP1-treated neurons, respectively. (i) Western blot of samples in g with TubΔCasp6 and full-length tubulin antibodies