Figure 3

CathD-deficient mice show reduced α-synuclein levels in detergent soluble brain extracts. Western blot analyses of fractionated whole brain homogenates from 24-day old, wild-type (+) and ctsd-/- mice (-) carried out under reducing conditions showing NP-40 (A) and SDS extracts (B). Immunoblots were probed with anti-aSyn Abs (Syn-1; mSA-1), anti-β-actin and anti-CathD Abs. Primary Ab detection was carried out with sheep anti-mouse and donkey anti-rabbit Ab, respectively. Under these immunoblotting conditions, variable amounts of the 16 kDa aSyn monomer (aSyn, FL) and traces of 12.5 kDa and 10 kDa (aSyn, ΔC) aSyn-reactive proteins are detectable; no reproducible > 150, 50 and 25 kDa aSyn-positive bands are seen.(C) ELISA [hSA2/Syn1-B] quantification of NP-40 and SDS extracts from ctsd wild-type (WT) and mutant ctsd-/- (KO) mouse brains and of snca WT and snca-/- (KO) lysates serving as positive and negative controls. Ctsd-genotyped brain extracts were loaded in triplicates at three different dilutions each (1:500 to 1:2,000) onto a 384-well plate and read at 405 nm, as described [39, 40]. Note, the absence of OD405 nm signals in snca KO mouse tissue. Bar graphs represent the mean group values (± SD) of mice that were individually analyzed (numbers as in A-C). The concentrations of total aSyn (in ng/μl) present in NP-40 extracts measured: ctsd WT, 1.34 ± 0.25; ctsd KO, 1.09 ± 0.26; and in the SDS fractions: ctsd WT, 0.60 ± 0.25; and ctsd KO, 0.48 ± 0.28.