Journal: PLOS ONE

Article Title: Human pericytes degrade diverse α-synuclein aggregates

doi: 10.1371/journal.pone.0277658

Figure Lengend Snippet: (A) Electron microscopy of α-syn strains before and after fragmentation. (B) Live cell imaging of fluorescently tagged α-syn for 30 hours. (C) Immunofluorescent labelling of α-syn strains after 30 min in control pericytes with α-syn epitope-specific antibody AA103-108. (D) Schematic representation of experimental setup. (E) Loading of western blots. Lane 1 only contains pure α-syn aggregate, whereas lane 2–5 shows α-syn isolated from pericytes. (F) Detailed fluorescent western blot with α-syn epitope-specific antibodies detecting Ribbons with α-syn antibody AA103-108 (magenta), AA124-134 (yellow) and merge showing overlap of α-syn bands. Various bands are identified indicating full length (14.4kDa, cyan arrow) and cleaved α-syn (red and blue arrows). α-syn detection on individual western blots for Fibrils, Ribbons, fibrils65, fibrils91, fibrils110 and no-treatment control (PBS) with α-syn epitope specific antibodies showing full length α-syn (cyan arrow) and cleaved α-syn fragments. Fibrils110 lacks a full-length band as aggregate is made up of C-term cleaved α-syn (green arrow). (G) AA 103–108, (H) AA124-134, and (H) GAPDH blot corresponds to blot shown in G after antibody stripping and relabelling. Full blots shown in images. Confocal image with orthogonal views showing pericyte with internalised α-syn Fibrils after 24 hours (cyan arrows, K). Scale bars represent 100 nm (A), 20 μm (B,C), 10 μm (K).

Article Snippet: Antibodies raised against α-syn C-terminus (Mouse anti α-syn, Amino Acid (AA) 124–134 (1:1,000) and ab1903 (Abcam); Mouse anti α-syn 4B12, AA 103–108, IgG1 (1:2,000, MA1-90346, ThermoFisher), GAPDH (1:1,000, ab9484, Abcam).

Techniques: Electron Microscopy, Live Cell Imaging, Western Blot, Isolation, Stripping Membranes

Journal: Nature Communications

Article Title: A reversible state of hypometabolism in a human cellular model of sporadic Parkinson’s disease

doi: 10.1038/s41467-023-42862-7

Figure Lengend Snippet: a Quantification of total mitochondrial mass and functional mitochondria in hNPCs or b DAns. Total mitochondrial mass was visualized using immunostainings with an antibody against the ATP synthase (ATP5F1A). Functional mitochondria with an active membrane potential were visualized using a MitoTracker probe (200 nM for 20 min). Images are exemplarily shown for hNPCs of clone i1E4-R1-003 (Ctrl), and iR66-R1-007 (sPD); for DAns of clone i1E4-R1-003 (mitoTracker—Ctrl), iR66-R1-007 (mitoTracker—sPD), i1JF-R1-018 (ATP5F1A—Ctrl), and iJ2C-R1-015 (ATP5F1A—sPD). Scale bar = 20 μm. c Violin plots highlight some morphological characteristics of mitochondria in hNPCs or d DAns: number of mitochondria (count), mitochondrial area, skeleton length, number of branch points, and mean intensity. On average, 112 (hNPCs—ATP5F1A), 160 (hNPCs—mitoTracker), 370 (DAns—ATP5F1A), 360 (DAns—mitoTracker) cells per clone were analyzed. e To assess alterations in the mitochondrial fusion machinery, expression of the mitofusions MFN1 , MFN2 , and OPA1 was quantified in hNPCs by RT-qPCR. Cells were cultivated on the energy substrates used in the Seahorse XF analysis (25 mM glucose or 5 mM pyruvate). f To assess alterations in the mitochondrial fission machinery, expression of DRP1 and its phosphorylation on Ser 616 was quantified in hNPCs by western blot. Protein levels were normalized to ACTB. g Western blots are exemplary shown for some hNPC clones. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value or to the most extreme data point which is no more than 1.5 times the interquartile range ( c , d ). Each dot represents one patient. n = 5 Ctrl and 7 sPD patient-derived cell clones, in triplicates. p- values were determined by linear mixed effects model ( c , d ); one-way ANOVA with Sidak’s Post hoc test ( p -values are provided together with the source data) ( e , f ). * p < 0.05; ** p < 0.01; *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies NDUFB8 (459210, Novex; 1:500), Complex II- Subunit 30 (SDHB) (459230, Thermo Fisher Scientific; 1:500), UQCRC2 (ab14745, Abcam; 1:2500), MT-CO2 (ab110258, Abcam; 1:1000), ATP5F1A (ab14748, Abcam; 1:4000), DRP1 (5391, Cell Signaling; 1:1000), DRP1 phospho-Ser616 (4494, Cell Signaling; 1:1000), TUBA (GTX628802, Genetex; 1:20,000), ACTB (ABO145-200, OriGene; 1:2000), OGDHL (17110-1-AP, Proteintech, 1:5000), GAPDH (GTX627408 peroxidase coupled, Genetex, 1:20,000), SLC2A1 (MA5-31960, Invitrogen, 1:5000), SLC2A3 (PA5-72331, Thermo Fisher Scientific, 1:5000) were incubated in blocking buffer overnight at 4 °C.

Techniques: Functional Assay, Membrane, Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Clone Assay, Derivative Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

doi: 10.3389/fcimb.2015.00067

Figure Lengend Snippet: Flow cytometry analyses for GAPDH expression on the surface of P. blaschkeae (SAG 2064), P. zopfii GT2 (SAG 2021), and P. zopfii GT1 (SAG 2063) . A representative image is shown. Cells in suspension were incubated with anti-GAPDH polyclonal antibodies, followed by incubation with Alexa-Fluor 488-labeled goat anti-rabbit IgG. As controls, cells were incubated only with the Alexa-Fluor 488-labeled goat anti-rabbit IgG to exclude any background. The experiment was repeated three independent times and results of one representative experiment are shown.

Article Snippet: Cells were incubated with 5–10 μg/ml polyclonal anti-GAPDH (Rockland, Limerick, USA) for 1 h at 37°C.

Techniques: Flow Cytometry, Expressing, Incubation, Labeling