Journal: The Journal of Biological Chemistry

Article Title: Structural analysis of the P132L disease mutation in caveolin-1 reveals its role in the assembly of oligomeric complexes

doi: 10.1016/j.jbc.2023.104574

Figure Lengend Snippet: P132L is recruited into DRMs by WT CAV1 in CAV1−/−MEFs. DRMs were isolated from CAV1−/− MEF cells transiently transfected with (A) CAV1-myc, (B) P132L-HA, or (C) CAV1-myc plus P132L-HA. Fractions were analyzed by SDS-PAGE/Western blotting. Flotillin-1 and calnexin were used as markers for DRMs and detergent soluble fractions, respectively. Fraction numbers are indicated at the top of the blots and the positions of the DRM fractions are indicated by red lines.

Article Snippet: Rabbit anti-CAV1 pAb (catalog number 610060), mouse anti-flotillin-1 mAb (catalog number 610820), and mouse anti-calnexin mouse mAb (catalog number 610523) were purchased from BD Transduction Laboratories.

Techniques: Isolation, Transfection, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: The AAA-ATPase VPS4 Regulates Extracellular Secretion and Lysosomal Targeting of α-Synuclein

doi: 10.1371/journal.pone.0029460

Figure Lengend Snippet: A . wt and A53T mutant αSYN were inducibly expressed in SH-SY5Y cells for 48 hours. Culture medium as well as whole cell lysates (50 µg protein per lane) were subjected to Western blot analysis. The collected media were further separated into the supernatant and exosome-containing pellets before loading onto gels. Alix, Hsp90, and BSA were used as markers for exosome, cytosol, and culture medium, respectively. In the neuronal culture medium, both monomeric/oligomeric wt and mutant αSYN were recovered in the supernatant ( dagger ) rather than exosome-containing pellets ( hash ). Asterisk indicates unspecific band. B . The resuspended exosome-containing pellets from the culture medium were further separated by sucrose-density gradient followed by Western blot analysis. Immunoblot probed with synuclein-1 anti-αSYN, anti-PrP Abs and the successful separation of exosome was confirmed by exosomal markers, Flotillin-1 and Alix. As shown in the blot, PrP migrated near the top of the density gradient (fraction #8) with concomitant enrichment of exosome-associated proteins. By contrast, no exosomal enrichment was observed with αSYN. C . CSF (1.5 mL) from 5 PD patients together with age-matched controls was pooled and exosome-containing pellets were isolated by successive centrifugation indicated. Equal concentrations (50 µg per lane) of total CSF samples were loaded alongside CSF-derived exosomes and then probed with anti-αSYN and PrP antibodies. PrP detected in CSF-derived exosomes was enriched compared to neat CSF. αSYN was weakly but specifically detected in neat CSF, whereas no αSYN-positive signal could be detected in CSF-derived exosomes. No significant difference was observed in the expression levels of CSF αSYN between PD patients and normal controls. Representative Western blots from three independent experiments are presented.

Article Snippet: After a blocking step with Tris-Buffer Saline (TBS: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.05% Tween 20 (TBST) supplemented with 5% nonfat dry milk, membranes were incubated with anti-cMyc mouse mAb (clone 9E10, 1∶1000; DSHB), M2 anti-FLAG/M2 (1∶1000; Sigma) mouse mAb, anti-GFP mouse mAb (1∶4000; MBL, Nagoya, Japan) anti-synuclein-1 mouse mAb (1∶1000; BD Bioscience, San Jose, CA), anti-Alix mouse mAb (clone 3A9, 1∶1000; CST), anti-flotillin-1 mouse mAb (1∶500; BD Transduction laboratories, Franklin Lakes, NJ), anti-Hsp90 mouse mAb (1∶4000; Stressgen, Victoria, BC, Canada), anti-BSA rabbit polyclonal antibody (pAb) (clone B-140, 1∶4000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-prion protein mouse mAb (1∶1000; Sigma), anti-ubiquitin Ab (clone P4D1, 1∶1000; Santa Cruz), anti-LAMP-1 mouse mAb (clone H4A3, 1∶1000, DSHB), anti-Rab5 rabbit pAb (1∶4000, Santa Cruz), and anti-Rab11 rabbit pAb (1∶1000; CST, Danvers, MA).

Techniques: Mutagenesis, Western Blot, Isolation, Centrifugation, Derivative Assay, Expressing