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, Control, Western Blot, Isolation, Stripping Membranes

Journal: Journal of Neuroscience

Article Title: Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection

doi: 10.1523/jneurosci.3200-07.2008

Figure Lengend Snippet: Figure9. p21actsinthecytoplasmtoprotectneuronsfromoxidativestress-induceddeath. A,Westernblotanalysistodetectrelativelevelsofp21innuclearandcytoplasmicfractionsfrom primarycorticalneuronalculturestreatedwithTSA(0.66M)inthepresenceorabsenceofHCA (5mM).ControlisnoTSAtreatment.RelativelevelsofGAPDHorNeuNandHistoneH3areshown to indicate purity of cytoplasmic or nuclear fractions, respectively. Antibodies against p21, GAPDH,NeuN,andHistoneH3wereused(seeMaterialsandMethods).B,Westernblotanalysis to detect relative levels of GFP, p21-GFP fusion protein or p21NLS-GFP fusion protein in cytoplasmic (C) and nuclear (N) fractions from HT22 murine hippocampal cells stably trans- fected with pEGFP, pEGFP-p21, or pEGFP-p21-NLS and treated with or without HCA (5 mM). Antibodies against p21 (top) or GFP (middle) were used (see Materials and Methods). The antibody for p21 does not detect the p21NLS-GFP fusion protein because the epitope this monoclonalantibodyrecognizesislocatedwithinthedeletednuclearlocalizationsignal(NLS). However, in addition to p21-GFP, this antibody detects endogenous p21, which is unchanged withHCAtreatment.C,GraphshowingviabilityofpEGFP-(darkgray),pEGFP-p21-(lightgray), and pEGFP-p21-NLS- (medium gray) transfected HT22 cells, as determined using the MTT assay,aftertreatmentwithincreasingconcentrationsofHCA(2.5–7.5mM)for24h.Graphbars depict mean SD. *Significant protection by p21 and p21-NLS relative to GFP control, p 0.001, by two-way ANOVA followed by Bonferroni posttests.

Article Snippet: Nonspecific binding was inhibited by incubation in Tris-buffered saline with Tween 20 (TBST: 50 mM Tris-HCl, pH 8.0, 0.9% NaCl, and 0.1% Tween 20) containing 5% nonfat milk for at least 1.5 h. Primary antibodies against p21 (BD Biosciences, San Jose, CA), p15 (Santa Cruz Biotechnology, Santa Cruz, CA), p16 (BD Biosciences), p27 (BD Biosciences), p57 (Millipore, Billerica, MA), pRb (BD Biosciences), GFP (Invitrogen), histone H4 (Millipore), acetyl histone H4 (Millipore), histone H3 (Millipore), phospho-JNK (Cell Signaling, Danvers, MA), total JNK (Cell Signaling), GAPDH (Millipore), NeuN (Millipore), -tubulin (Sigma-Aldrich), and HA (Sigma-Aldrich) were diluted in TBST containing 5% milk overnight at 4°C followed by incubation with respective horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) for 2 h at room temperature.

Techniques: Stable Transfection, Transfection, MTT Assay, Control

Journal: Scientific Reports

Article Title: Impact of Escherichia coli K12 and O18:K1 on human platelets: Differential effects on platelet activation, RNAs and proteins

doi: 10.1038/s41598-018-34473-w

Figure Lengend Snippet: Western blot and ELISA analysis of HMBS and ATP2C1. ( a ) Cell lysates were applied to SDS-PAGE gels under reducing conditions. HMBS and ATP2C1 protein were detected in platelets using a polyclonal antibody. Incubation of platelets with E. coli K12 and E. coli O18:K1 in 1:5 or 1:10 platelet-bacteria ratios converted HMBS 47 kDa form to a 40 kDa protein. ATP2C1 was not affected. GAPDH was used as a loading control. ( b ) The releasates of the platelet-bacteria mix were collected after centrifugation (500 g, 10 minutes without break). HMBS, ATP2C1 and LRCH4 levels were measured by ELISA. Data represents the mean of three independent experiments (n = 3). ATP2C1 was detectable in platelet supernatants, while HMBS and LRCH4 proteins were either not released from platelets or in concentrations below the detection level of the ELISA (data not shown). BDL, below detection limit. Western blot results are representative image of three replications. The same exposure was applied equally across the entire image. The original pictures of the full-length western blots can be found in Supplementary Fig. .

Article Snippet: The blots were incubated overnight with primary anti-human antibodies (rabbit polyclonal ATP2C1 (1:750), LRCH4 (1:500), HMBS (1:1000) or GAPDH (1:600) from Atlas Antibodies, Bromma, Sweden).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, SDS Page, Incubation, Bacteria, Control, Centrifugation

Journal: iScience

Article Title: An antisense-long-noncoding-RNA modulates p75 NTR expression levels during neuronal polarization

doi: 10.1016/j.isci.2024.111566

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal antibody anti-GAPDH , Cusabio , CatCSB-RA009232A0HU.

Techniques: Recombinant, Rapid Amplification of cDNA Ends, Sequencing, shRNA, Plasmid Preparation, Cloning, Software