mouse pan ubiquitin antibody  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: α-Synuclein aggregates inhibit ESCRT-III through sequestration and collateral degradation

doi: 10.1101/2025.01.13.632710

Figure Lengend Snippet: (A) Schematic describing the ESCRT reporter assay. An EGFP-RNF152 fusion is expressed alongside an IRES-driven mScarlet in the HEK α-syn expressing cell line. The mScarlet serves as an internal control for reporter expression, while the EGFP level tracks degradation of the RNF152 reporter, which is internalized into the lysosomal lumen and degraded in an ESCRT-III-dependent manner. EGFP, enhanced green fluorescent protein. IRES, internal ribosome entry site. (B) Representative immunoblot of lysates from cells expressing the ESCRT reporter with strong knockdown of CHMP2A and CHMP2B. Membranes were stained with anti-GFP, anti-CHMP2A, anti-CHMP2B, anti-α-syn, and anti-β-actin. β-actin served as a loading control. Quantification appears in Figure S3D (n=3). KD, knockdown. NT, non-targeting control. (C) Flow cytometry to measure the stability (EGFP/mScarlet) of the ESCRT reporter after strong CHMP2A and CHMP2B knockdown, normalized to the value in NT. Error bars indicate mean ± SEM (n=3). ***p<0.001 relative to NT control by one-way ANOVA. (D) Flow cytometry of the ESCRT reporter cell line after exposure to PFFs, Lpf, and/or Dox to control the α-syn aggregation state. Measurements are normalized to the untreated condition. Error bars signify mean ± SEM (n=5). ****p<0.0001 relative to untreated control by two-way ANOVA. (E) Flow cytometric ESCRT reporter stability measurements of the effect of combining aggregation induction (Lpf/PFF vs Lpf) with either mild CHMP2A/B knockdown or 5 hr exposure to 100 nM of the E1 ubiquitin ligase inhibitor MLN7243 (MLN) (n=3 for the knockdown conditions and n = 5 for MLN conditions). EGFP/mScarlet values are normalized to appropriate control condition, either Lpf/NT or Lpf/DMSO. Error bars denote mean ± SEM. Arrows mark the EGFP/mScarlet ratios that would be expected if the combined perturbations were additive. Numbering for each bar (referred to in the text) appears below. The supporting immunoblot of the mild knockdown and a quantification thereof appear in Figures S3I and S3J. **p<0.01;****p<0.0001 relative to NT or DMSO control by two-way ANOVA. See also Figure S3.

Article Snippet: 1 mg of lysate was incubated with 50 µL of pre-equilibrated Ubiquitin pan-selector resin (NanoTag Biotechnologies, N2510) for 1 hr at 4°C with rotation.

Techniques: Reporter Assay, Expressing, Control, Western Blot, Knockdown, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: α-Synuclein aggregates inhibit ESCRT-III through sequestration and collateral degradation

doi: 10.1101/2025.01.13.632710

Figure Lengend Snippet: (A and B) Representative immunoblots of HEK α-syn expressing cell lysates after treatment with either PBS or PFFs (without Lpf) for increasing amounts of time (A) or for 6 days with Dox treatment at different times (B), according to the timeline above. Note in (B) that 6 days of Dox treatment indicates Dox addition on day 0, with 5 days of treatment indicating addition on day 1, etc. Immunoblots were stained with anti-α-syn, anti-CHMP2B, and anti-β-actin antibodies. β-Actin was the loading control. Densiometric quantifications of the immunoblots appear below. CHMP2B values are normalized to the value obtained at the first timepoint and α-syn values are normalized to the maximum value – 6 d in (A) and 0 d in (B). Error bars signify mean ± SEM (n=3). *p<0.05;***p<0.001;****p<0.0001 by two-way ANOVA, pairwise comparisons shown between PBS/PFF-treated CHMP2B (blue) or high MW α-syn (red) measurements at matched timepoints. (C) Densiometric quantification of immunoblots in Figure S4C, showing the effect of 8 hr inhibitor treatment on CHMP2B levels with and without aggregation induction. Bz, 500 nM bortezomib (proteasome inhibitor). MLN, 500 nM MLN7243 (E1 ubiquitin ligase inhibitor). Baf, 250 nM bafilomycin A1 (lysosomal V-ATPase inhibitor). Data are presented after β-actin normalization and subsequent normalization to the Lpf/DMSO control condition. Error bars indicate mean ± SEM (n=3). *p<0.05 by two-way ANOVA. Only significant comparisons are shown. (D) Densiometric quantification of immunoblots in Figure S4E, testing how aggregation induction affects transfected FLAG-tagged CHMP2B variant levels. CHMP2B levels were normalized to β-actin, then to the value obtained for each variant in the Lpf control condition. Error bars represent mean ± SEM (n=5). n.s. p>0.05;*p<0.05 by two-way ANOVA. See also Figure S4.

Article Snippet: 1 mg of lysate was incubated with 50 µL of pre-equilibrated Ubiquitin pan-selector resin (NanoTag Biotechnologies, N2510) for 1 hr at 4°C with rotation.

Techniques: Western Blot, Expressing, Staining, Control, Transfection, Variant Assay