Journal: Current biology : CB

Article Title: Baculovirus actin-based motility drives nuclear envelope disruption and nuclear egress

doi: 10.1016/j.cub.2018.05.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: mouse anti-tubulin (clone E7) , Developmental Studies Hybridoma Bank, University of Iowa , RRID: AB_2315513.

Techniques: Virus, Recombinant, Cell Culture, Plasmid Preparation, Software

Journal: Brain : a journal of neurology

Article Title: Changes in readthrough acetylcholinesterase expression modulate amyloid-beta pathology.

doi: 10.1093/brain/awm276

Figure Lengend Snippet: Fig. 6 AChE-R protein levels are reduced in the hippocampus of Alzheimer’s disease patients. (A) Total AChE activity was mea- sured in hippocampal homogenates from 10 Alzheimer’s disease patients and five non-demented controls (NDC). (B) Total AChE immuno-blot of hippocampal homogenates from NDCs and Alzheimer’s disease patients. A 30% decrease in labelling intensity of intact AChE is observed, similar to that seen in AChE activity. Note the presence of shorter degradation products (filled circle). Scheme: Total AChE was immuno-detected by an antibody that captures all AChE variants (H134). (C) A specific antibody against AChE-R (anti-R) detected reduction to 20% of NDC levels in AChE-R protein levels from hippocampal homogenates of Alzheimer’s disease patients (P50.01, Mann^Whitney test). R marks recombinant AChE-R positive control. -tubulin labelling is shown as loading control in both (B) and (C). Note the Alzheimer’s disease-associated increase in AChE-R degredation products (filled circle) migrating similarly to those labelled with the H134 antibody.

Article Snippet: Immuno-blot analysis of human hippocampal homogenate samples involved rabbit anti-human AChE-R antibodies directed at the unique C-terminus of AChE-R (1:700) (Sternfeld et al., 2000), anti total AChE (H134, Santa Cruz, Santa Cruz, CA, 1:700) and anti -tubulin (Santa Cruz, 1:2000).

Techniques: Activity Assay, MANN-WHITNEY, Recombinant, Positive Control, Control

Journal: iScience

Article Title: Derived myeloid lineage induced pluripotent stem as a platform to study human C-C chemokine receptor type 5Δ32 homozygotes

doi: 10.1016/j.isci.2023.108331

Figure Lengend Snippet:

Article Snippet: After 24 hours of recovery in Essential 8TM Medium (Gibco), in order to drive cells into mesoderm progenitor differentiation, cells were allowed to grow in chemically defined differentiation medium (MDM) containing Iscove’s modified Dulbecco’s medium (IMDM, Invitrogen) supplemented with Ham's F-12 Nutrient Mix, GlutaMAXTM supplement (Invitrogen) at 1:1 ratio, Albucult (recombinant human - rh Albumin) (5 mg/ml; Novozymes Delta), a-Monothioglycerol (a-MTG) (3.9 ml per 100 ml; Sigma-Aldrich), Protein-free hybridoma mixture II (PFHMII) (5%; Invitrogen), Ascorbic acid 2 phosphate (50 μg/ml; Sigma-Aldrich), Penicillin/streptomycin (50 U Pen G/50 mg streptomycin sulfate; Invitrogen), Insulin Transferrin Selenium-X Supplement (Invitrogen).

Techniques: Virus, Recombinant, Modification, Purification, Blocking Assay, Control, Library Quantification, Luminex, Pore Size, Amplification, Software

Journal: Neuron

Article Title: A Selectivity Filter Gate Controls Voltage-Gated Calcium Channel Calcium-Dependent Inactivation

doi: 10.1016/j.neuron.2019.01.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains Dh5α competent E. coli MCLAB Cat#DA-196 Chemicals, Peptides, and Recombinant Proteins ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) Sigma-Aldrich Cat#E4378 1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis (BAPTA) Santa Cruz Biotechnology Cat#sc-202076 Dulbecco’s modified Eagle’s medium (DMEM) GIBCO Cat#11965-092 fetal bovine serum (FBS) GIBCO Cat#16140-071 L-glutamine GIBCO Cat#25030-081 penicillin/streptomycin UCSF Cell Culture Facility Cat#CCFGK004-153K01 Lipofectamine 2000 Invitrogen Cat#11668-019 Matrigel BD Biosciences Cat#354234 Critical Commercial Assays T7 mMessenger kit Thermo Fisher Scientific Cat# AM1344 QuikChange Site-Directed Mutagenesis Kit Agilent Cat#200515 Experimental Models: Cell Lines HEK293 ATCC Cat#CRL-1573 Xenopus oocytes This study N/A Experimental Models: Organisms/Strains Xenopus Laevis Nasco Cat# LM00531 Recombinant DNA human Ca V 1.2/pcDNA3.1 Findeisen and Minor, 2009 N/A human Ca V 1.2 D707A/pcDNA3.1 Shaya et al., 2014 N/A human Ca V 1.2 D707N/pcDNA3.1 Shaya et al., 2014 N/A human Ca V 1.2 D707G/pcDNA3.1 Shaya et al., 2014 N/A human Ca V 1.2 D707E/pcDNA3.1 This study N/A human Ca V 1.2 E1115A/pcDNA3.1 This study N/A human Ca V 1.2 D367A/pcDNA3.1 This study N/A human Ca V 1.2 E1119A/pcDNA3.1 This study N/A human Ca V 1.2 D1420A/pcDNA3.1 This study N/A human Ca V 1.2 G364D/D707G/pcDNA3.1 This study N/A human Ca V 1.2 D707G/G1116D/pcDNA3.1 This study N/A human Ca V 1.2 D707A/A1417D/pcDNA3.1 This study N/A rat Ca V 1.3 42a /pcDNA6/V5-His ABC Xu and Lipscombe, 2001 Addgene#26577 rat Ca V 1.3 D726A/pcDNA6/V5-His ABC This study N/A rat Ca V 1.3 E1121A/pcDNA6/V5-His ABC This study N/A human Ca V 2.1/pcDNA3.1 Kim et al., 2008 N/A human Ca V 2.1 D667A/pcDNA3.1 This study N/A human Ca V 2.1 E1461A/pcDNA3.1 This study N/A rat Ca V β 2a /pTracer-CMV2-GFP Findeisen and Minor, 2009 N/A rabbit Ca V β 3 /pSport This study N/A rabbit Ca V α 2 δ-1/pcDNA3.1 Findeisen and Minor, 2009 N/A sv40 T-antigen/Bluescribe addgene Cat#21826 Software and Algorithms pCLAMP 9 Molecular Devices N/A Clampfit 10 Molecular Devices N/A SigmaStat 3.1 Systat N/A Open in a separate window KEY RESOURCES TABLE Ca V selectivity filter forms the calcium-dependent inactivation (CDI) endpoint Conserved Ca V domain II selectivity filter (+1) aspartate plays an active role in CDI Ca V selectivity filter asymmetry is important for CDI Ca V s gating relies on an SF-based gating framework shared among the VGIC superfamily

Techniques: Recombinant, Modification, Cell Culture, Mutagenesis, Software

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit

doi: 10.1074/jbc.M117.809301

Figure Lengend Snippet: Toxoplasma strains used in this study

Article Snippet: Parasite manipulations Strain RHΔ ku80 Δ hxgprt (RHΔΔ) of T. gondii was cultured on preformed monolayers of human foreskin fibroblasts (ATCC catalog no. SCRC-1041), or BJ fibroblasts (ATCC catalog no. CRL-2522) if indicated, in the presence of Complete Medium, which consisted of DMEM (Corning Inc.) supplemented with 10% (v/v) FBS, 2 m m l -glutamine, and 100 units/ml penicillin/streptomycin (Corning) at 37 °C in a humidified CO 2 (5%) incubator.

Techniques: Selection, Marker

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit

doi: 10.1074/jbc.M117.809301

Figure Lengend Snippet: glt1Δ parasites accumulate the trisaccharide form of Skp1. A, Western blot analysis of Skp1 from equivalent numbers (3 × 106 cells) of parental RHΔΔ, phyAΔ, glt1Δ, or complemented cells. Soluble S16 fractions were separated on a 4–12% SDS-polyacrylamide gel, electroblotted, and probed with anti-TgSkp1 (UOK75) antiserum. B–E, analysis of the Skp1 glycosylation by mass spectrometry, from RHΔΔ (left half) and glt1Δ (right half) cells. Skp1 was enriched by immunoprecipitation and converted to peptides with trypsin. Total peptides were analyzed by nanoLC/MS-MS (note that retention times varied between the left- and right-hand trials, which were conducted at different times). The vertical dashed boxes show approximate elution intervals for glycoforms of the Pro-154–containing peptides from Skp1. Spectral counts corresponding to exact m/z matches that appear as both doubly and triply charged forms are annotated at the side. B, extracted ion scans from full-ion mode for the unmodified peptide containing Pro-154, showing scans for [M + 2H]2+ and [M + 3H]3+ ions. C, extracted ion scans for corresponding to the HexHexNAc- derivative of the Pro-hydroxylated peptide. D, same for the dHexHexHexNAc- derivative. E, same for the fully glycosylated Hex2dHexHexHexNAc- derivative. No other glycoforms were detected.

Article Snippet: Parasite manipulations Strain RHΔ ku80 Δ hxgprt (RHΔΔ) of T. gondii was cultured on preformed monolayers of human foreskin fibroblasts (ATCC catalog no. SCRC-1041), or BJ fibroblasts (ATCC catalog no. CRL-2522) if indicated, in the presence of Complete Medium, which consisted of DMEM (Corning Inc.) supplemented with 10% (v/v) FBS, 2 m m l -glutamine, and 100 units/ml penicillin/streptomycin (Corning) at 37 °C in a humidified CO 2 (5%) incubator.

Techniques: Western Blot, Glycoproteomics, Mass Spectrometry, Immunoprecipitation, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit

doi: 10.1074/jbc.M117.809301

Figure Lengend Snippet: Toxoplasma extracts express glt1-dependent Skp1 glucosyltransferase activity. A, desalted cytosolic extracts were assayed for the ability to transfer 3H from UDP-[3H]Gal or UDP-[3H]Glc to FGGn-Skp1, based on incorporation into the Skp1 band after SDS-PAGE. Normal (RHΔΔ) and glt1Δ mutant extracts were assayed for 1.5 or 3 h as indicated. Negligible incorporation was detected in the rest of the SDS-PAGE lane (data not shown). These data, which were collected in parallel on the same extracts, are representative of other trials conducted under varied conditions (data not shown). B, after electroblotting from the SDS-polyacrylamide gel to a polyvinylidene difluoride membrane and staining with Ponceau, the Skp1 band and surrounding regions were separately acid hydrolyzed and subjected to high pH anion exchange chromatography on a Dionex PA-1 column in the presence of internal standards. Monosaccharide elution was monitored by pulsed amperometric detection (nC), and fractions were collected for scintillation counting for 3H. Similar results were obtained for UDP-[3H]Gal (shown) and UDP-[3H]Glc reaction products (data not shown).

Article Snippet: Parasite manipulations Strain RHΔ ku80 Δ hxgprt (RHΔΔ) of T. gondii was cultured on preformed monolayers of human foreskin fibroblasts (ATCC catalog no. SCRC-1041), or BJ fibroblasts (ATCC catalog no. CRL-2522) if indicated, in the presence of Complete Medium, which consisted of DMEM (Corning Inc.) supplemented with 10% (v/v) FBS, 2 m m l -glutamine, and 100 units/ml penicillin/streptomycin (Corning) at 37 °C in a humidified CO 2 (5%) incubator.

Techniques: Activity Assay, SDS Page, Mutagenesis, Membrane, Staining, Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit

doi: 10.1074/jbc.M117.809301

Figure Lengend Snippet: Substrate specificities of His6-Glt1. A, comparison of glycosyl donor specificities, based on UDP-sugar hydrolysis. Recombinant Glt1 was incubated with each UDP-sugar at 50 μm, and released UDP was detected using the UDP-GloTM assay. Data are representative of three trials, and error bars represent S.D. for one reaction conducted in triplicate. B, comparison of utilization of UDP-Glc or GDP-Man as donors in a transferase assay toward 1.5 mm FG-Bn, which represents the non-reducing terminal disaccharide of Skp1, using a UDP or GDP (NDP) detection assay. S.D. values of three technical replicates are shown. C, dependence of transferase activity on UDP-Glc concentration, using 2 mm FG-Bn as the acceptor substrate in reactions monitored by incorporation of 3H from UDP-[3H]Glc. Data represent the mean of two technical replicates ± S.D. The Km calculation is based on the Michaelis–Menten model. D, time dependence of reactions comparing FG-pNP with FGGn-Bn, which models the full Skp1 trisaccharide, assayed as in C. The acceptor and donor substrates were 2 mm and 2 μm, respectively. Error bars, S.D. between duplicate samples. E, comparison of different potential acceptor substrates, as in D, in reactions incubated for 2 h. F, dependence of transferase activity on FGGn-pNP concentration, in the presence of 40 μm UDP-Glc. Data are representative of two independent experiments, and the kinetic calculations are based on the Michaelis–Menten model. G, dependence on Toxoplasma FGGn-Skp1 concentration, as in F. H, biochemical complementation of parasite extracts with His6-Glt1. Transferase reactions were conducted using UDP-[3H]Glc as the donor and desalted S100 extracts of parental (RHΔΔ) or glt1Δ extracts as the acceptor in the presence or absence of His6-Glt1. Samples were separated by SDS-PAGE, and, after fixation, gel lanes were sliced from top to bottom. Incorporation was determined by scintillation counting.

Article Snippet: Parasite manipulations Strain RHΔ ku80 Δ hxgprt (RHΔΔ) of T. gondii was cultured on preformed monolayers of human foreskin fibroblasts (ATCC catalog no. SCRC-1041), or BJ fibroblasts (ATCC catalog no. CRL-2522) if indicated, in the presence of Complete Medium, which consisted of DMEM (Corning Inc.) supplemented with 10% (v/v) FBS, 2 m m l -glutamine, and 100 units/ml penicillin/streptomycin (Corning) at 37 °C in a humidified CO 2 (5%) incubator.

Techniques: Comparison, Recombinant, Incubation, Glutathione S-Transferase Assay, Detection Assay, Activity Assay, Concentration Assay, SDS Page