Fig. 4.

SPOP knockdown protects against DPR toxicity in mammalian primary neurons without reducing the abundance of known SPOP ubiquitination targets or DPRs themselves. (A) Cortical neuron cultures were infected with HSV engineered to express LacZ, a scrambled sequence miR or two different miRs targeting SPOP. Endogenous neuronal SPOP levels are reduced by targeted miRs. (B) DIV 14 spinal cord neuron cultures were coinfected with HSV engineered to express the indicated DPR and either the scrambled sequence miR (control, “cont”) or miR targeting SPOP. Control cultures were infected with HSV-LacZ. Cultures were processed for immunocytochemistry and motor neuron counts were obtained 5 d later. By ANOVA, DPRs lead to a statistically significant reduction in motor neuron number (compared with LacZ expressing cultures) and knockdown of SPOP leads to a statistically significant protection against SPR toxicity (e.g., GA50 group, F_(3,44) = 72.49, P < 0.001; GR50 group, F_(3,43) = 204.7, P < 0.0001; PR50 group, F_(3,44) = 100.8, P < 0.0001). Pairwise comparisons utilized Tukey’s post hoc multiple comparisons test. ****P < 0.0001. (C) Cortical neuron cultures were infected with HSV engineered to express LacZ or the GA50, PR50, or GR50 DPRs. Simultaneously, cultures were infected with either the HSV engineered to express the scrambled sequence miR or SPOP miR. Then, 2 d later, before any neuron loss is detected, lysates were probed for known SPOP targets. No statistically significant group differences were detected in band intensities by ANOVA (SI Appendix, Fig. S7). (D) Lysates from experiments described in C were probed for GA, GR, or PR and imaged with short or long exposures (to see less abundant higher molecular weight species). Putative monomers are noted with “>.” No statistically significant group differences were detected in scan band intensities by ANOVA (SI Appendix, Fig. S8).