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Figure 1. Western blot analysis of Proteasome 19S Rpn10/S5a subunit, mAb (S5a-18) (Prod. No. BML-PW9250): Lane 1: MW marker, Lane 2: 3T3 (Prod. No. ADI-LYC-3T100), Lane 3: L929, Lane 4: PC-12 (Prod. No. ADI-LYC-PC100), Lane 5: MDBK, Lane 6: HeLa S100 fraction (Prod. No. BML-SW8750).
Lysosomal‐driven deregulation of α‐synuclein and CSPα degradationWB analysis of LC3 and p62 (an autophagy substrate) was performed on WT and MPS‐IIIA brain samples at the indicated ages. WB quantitation is shown.α‐Synuclein was immunoblotted in WT and MPS‐IIIA in both total and synaptosomal brain fractions at the indicated ages after sequential extraction with detergents with increased strength. Soluble (Sol.), lowly insoluble (L. Insol.), and highly insoluble (H. Insol.) forms correspond to the protein solubilized in Triton X‐100, 10% SDS and 8 M urea, respectively.Co‐immunofluorescence analysis of α‐synuclein with SMI‐32 in WT and MPS‐IIIA hippocampal neurons (DIV14). α‐Synuclein synaptic puncta present in a neurite tract of 10 μm is shown in a representative enlarged image. Quantification of α‐synuclein synaptic puncta was calculated from 30 different enlarged images.Confocal analysis of α‐synuclein (green) and LAMP1 (red) in WT and MPS‐IIIA hippocampal neurons (DIV14). Enlarged merge images are also shown. Co‐localization was quantified using the MCC (ImageJ) and displayed as percentage (MCC × 100) of α‐synuclein co‐localizing with LAMP1 (15 different images taken from 4 to 5 coverslips for each group).CSPα was immunoblotted in WT and MPS‐IIIA total brain lysates at the indicated ages after sequential extraction with detergents with increased strength as in (B).Co‐immunofluorescence analysis of CSPα and SMI‐32 in DIV14 hippocampal neurons. CSPα synaptic puncta was quantified as in (C).CSPα protein levels were evaluated by immunoblot analysis in WT and MPS‐IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as percentage of remaining protein at T0 (100%). The proteasome was inhibited as indicated. WB quantification is shown.Palmitoylation‐dependent shift in the molecular weight of CSPα was evaluated in WT and MPS‐IIIA brain samples at the indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulfhydryl agents (β‐mercaptoethanol or dithiothreitol).The protein levels of RPN10 (the regulatory subunit of 26S proteasome) and ubiquitinated proteins formed by Lys48 (K48) residue linkage (involved in protein degradation via the proteasome) were evaluated in WT and MPS‐IIIA brain samples at the indicated ages by WB analysis. WB quantitation is shown.Proteasome activity was evaluated by measuring the chymotrypsin‐like activity in WT and MPS‐IIIA mouse brain samples at different ages. Proteasome activity was expressed as percentage of WT activity.Data information: Data are means ± s.e.m.; N = 3 (biological triplicate) in WB quantitations. *P < 0.05, **P < 0.001, Student's t‐test: MPS‐IIIA at each age vs. WT (A, I, J); MPS‐IIIA vs. WT (C, D, F); either WT or MPS‐IIIA + prot. inhib. (at each time point) vs. MPS‐IIIA (G). Scale bars: 10 μm (C, D, F).Source data are available online for this figure.
Image collected and cropped by CiteAb under a CC-BY license from the following publication: Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases. EMBO Mol Med (2017)
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RUO – Research Use Only |
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