Figure S7.

UBQLN2 Is Aggregated in Hippocampus and Associates to HSP70 and HTT Aggregates; Characterization of UBQLN Antibodies, Related to Figures 1, 2, 4, 5, and 6

(A and B) Hippocampal or cortical extracts were examined from WT or P520T knock-in mice. UBQLN2 expression levels were quantified and found to be indistinguishable between brain regions in either genotype.

(C) UBQLN2 was found in the pellet fraction of the hippocampus in P520T knock-in, but not WT mice, in three additional independent pairs of animals.

(D) UBQLN2 co-localizes with HTT inclusions in R6/2 brains. Sections of 14 week R6/2 brains were stained for HTT (MW8 antibody) and UBQLN2.

(E) UBQLN2 was immunoprecipitated from 14-week-old R6/2 brains and blotted for the indicated proteins. HTT and HSP70 were detected in the stacking gel, indicated UBQLN2 interacts with SDS-insoluble HTT aggregates that are positive for HSP70.

(F–I) Validation of specificity for UBQLN antibodies produced in-house. All antibodies were raised in sheep. UBQLN1, UBQLN2 or UBQLN4 were knocked down using siRNA and the indicated antibody used for detection. No cross-reactivity between ubiquilins was seen.

(J) Purified untagged mouse UBQLN1, 2, 3 and 4 was further used to assess specificity of the raised antibodies, which confirm that there is no cross reactions.