goat anti mouse catb  (R&D Systems)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Wild-type GBA1 increases the α-synuclein tetramer–monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice

doi: 10.1073/pnas.2103425118

Figure Lengend Snippet: Hypothetical pathway of shifting native αS multimers toward excess monomers and therapeutic approaches against the buildup of lipid-rich and lysosomal foci. Lysosomal dysfunction—by GCase deficiency or other autophagy perturbations—induced by excess αS monomers at membranes, decreases CatB activity (a risk factor of PD penetrance in hu GBA1 carriers that can strip off perilipins), and this, in turn, yields to Plin-stabilized LDs unavailable for cytosolic degradation. The resultant, nutritional stress stimulates the production of new LDs, and their abnormal membrane expansion is detected by CCT-α [as in Gaucher’s Disease neuropathology ]. The relative abundance of cytosolic LDs retains TFEB in the cytosol (cyt) versus translocation to the nucleus (nucl), critical for lysosomal biogenesis and thereby lipid degradation ( , , ). The treatment with an SCD inhibitor (“5b”) likely acts upstream of this vicious cycle by decreasing unsaturated FAs that are typically stored in form of TAGs, the major component of LDs, thereby reducing the abundance of lipidic material. Alternatively, but not mutually exclusive, a relative increase in SFAs (by SCD inhibitor or increased GCase activity) can produce higher-order lipid domains in membranes and thereby create an αS- (and other protein) repellant surface, increasing the solubility of αS monomers that assemble dynamically into physiological αS tetramers and subsequently decrease the vesicle- and lipid-rich aggregates. Finally, certain SFAs can stabilize the helical structure of αS and thereby the tetramer formation. For additional references of the hypothesized pathway, see Results and Discussion and our previous publication .

Article Snippet: For immunofluorescent experiments, sections were blocked in 10% normal donkey serum and incubated overnight at 4 °C with abs to hu or tGCase (ab55080, 1:100; Abcam), LAMP1 (ab25245, 1:500; Abcam), anti-phosphorylated (pS129) αS (ab51253, 1:4,000; Abcam), goat CatB (RD Biosystems, 1:1,000), CatD (MAB 1029; RD Biosystems, 1:1,000), perilipin 2 (sc390169, Santa Cruz; 1:1,000), and TFEB (A700-070, 1:200; Bethyl Laboratories).

Techniques: Activity Assay, Stripping Membranes, Translocation Assay, Solubility

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Wild-type GBA1 increases the α-synuclein tetramer–monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice

doi: 10.1073/pnas.2103425118

Figure Lengend Snippet: wtGBA1 increases lysosomal enzyme maturation and activity. The sizeable LAMP1+ foci (FIJI “Maxima” plugin) lacked tGCase reactivity in 3K (magnified on the Right ), and GBA1 induction increased the colocalization of tGCase with more finely distributed LAMP1 puncta ( A ) and quantitation of colocalized puncta ( B ). ( C ) GCase activity measured in the cortex of GBA or vector-injected 3K and non-Ntg littermates at 6-mo postinjection using the 4MUG assay. Increased CatB immunoreactivity in GBA versus vec-3K ( D ) and quantification ( E ). ( F ) WB shows preforms of CatD (50 kDA) and CatB (43 to 46 kDA) and their LMW (CatD at 14 + 34 kDa and CatB at 25 to 26 kDa) products, representing cleavage at low pH+, consistent with increased CatB immunoreactivity and colocalization with LAMP1+ shown in D . ( G ) Quantification of the ratio between the higher and lower molecular signals of CatD and CatB. For histological analyses, n = 7 to 10 fields of three hippocampal sections from each genotype ( n = 3 to 4 each cohort). IOD, integrated optic density; col, colocalization; exp, exposure. Data are expressed as means ± SEM; * P < 0.05 and ** P < 0.01; two-way ANOVA, Tukey’s post hoc test, or unpaired two-tailed t test. (Scale bars, 20 µm.)

Article Snippet: For immunofluorescent experiments, sections were blocked in 10% normal donkey serum and incubated overnight at 4 °C with abs to hu or tGCase (ab55080, 1:100; Abcam), LAMP1 (ab25245, 1:500; Abcam), anti-phosphorylated (pS129) αS (ab51253, 1:4,000; Abcam), goat CatB (RD Biosystems, 1:1,000), CatD (MAB 1029; RD Biosystems, 1:1,000), perilipin 2 (sc390169, Santa Cruz; 1:1,000), and TFEB (A700-070, 1:200; Bethyl Laboratories).

Techniques: Activity Assay, Quantitation Assay, Plasmid Preparation, Injection, Two Tailed Test