Journal: International Journal of Molecular Sciences

Article Title: Expanding the Therapeutic Window of EGFR-Targeted PE24 Immunotoxin for EGFR-Overexpressing Cancers by Tailoring the EGFR Binding Affinity

doi: 10.3390/ijms232415820

Figure Lengend Snippet: Binding kinetics of the interactions between either soluble anti-EGFR monobody variants or PE24-fused ER-PE24 IT variants and immobilized human EGFR-ECD, as measured by bio-layer interferometry.

Article Snippet: The 96-well EIA/RIA plates (Corning, New York, NY, USA) were coated for 1 h at 25 °C with 250 μg/well (5 μg/mL, 50 μL) of the recombinant human EGFR-ECD (Sino Biological, 10001-H08H), mouse EGFR-ECD (Sino Biological, 51091-M08H), double-stranded DNA (1 μg/mL, Sigma-Aldrich, St. Louis, MI, USA; D4522;), insulin (5 μg/mL, Sigma-Aldrich, I9278;), cardiolipin (50 μg/mL, Sigma-Aldrich, C0563;), or hemocyanin (5 μg/mL, Sigma-Aldrich, H8283;) and blocked using the blocking solution [PBST (PBS, 0.05% ( v/v ) Tween-20, pH 7.4), 3% ( w/v ) skim milk].

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Expanding the Therapeutic Window of EGFR-Targeted PE24 Immunotoxin for EGFR-Overexpressing Cancers by Tailoring the EGFR Binding Affinity

doi: 10.3390/ijms232415820

Figure Lengend Snippet: Generation and characterization of ER-PE24 IT variants with different affinities for EGFR. ( A ) Schematic view of the ER-PE24 IT construction (lower panel), and predicted structure via AlphaFold (AlphaFold v.2.0) (upper panel). The anti-EGFR monobody (Green) was fused to the N-terminus of the B-cell epitope-removed PE24 toxin, LO10 PE24 (Cyan), via a GGS linker. ( B ) SDS-PAGE analyses of the purified ER-PE24 ITs (each 5 μg loading) under non-reducing conditions. The gel was stained with Coomassie Blue R-250. ( C ) Representative binding isotherms of the immobilized EGFR-ECD-Fc in relation to soluble ER-PE24 IT, as measured by bio-layer interferometry. The concentrations of ER-PE24 IT analyzed are indicated using different colors. The binding kinetics parameters are listed in . ( D ) Concentration-dependent binding activities of ER-PE24 ITs to human or murine EGFR-ECD, as determined by ELISA. Error bars represent the mean ± SEM ( n = 3).

Article Snippet: The 96-well EIA/RIA plates (Corning, New York, NY, USA) were coated for 1 h at 25 °C with 250 μg/well (5 μg/mL, 50 μL) of the recombinant human EGFR-ECD (Sino Biological, 10001-H08H), mouse EGFR-ECD (Sino Biological, 51091-M08H), double-stranded DNA (1 μg/mL, Sigma-Aldrich, St. Louis, MI, USA; D4522;), insulin (5 μg/mL, Sigma-Aldrich, I9278;), cardiolipin (50 μg/mL, Sigma-Aldrich, C0563;), or hemocyanin (5 μg/mL, Sigma-Aldrich, H8283;) and blocked using the blocking solution [PBST (PBS, 0.05% ( v/v ) Tween-20, pH 7.4), 3% ( w/v ) skim milk].

Techniques: SDS Page, Purification, Staining, Binding Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature neuroscience

Article Title: A glycolytic shift in Schwann cells supports injured axons

doi: 10.1038/s41593-020-0689-4

Figure Lengend Snippet: a, Top: Scheme of metabolomic analysis using extracts from nerve segments. Bottom: Concentrations of key energy metabolism intermediates in control and axotomized nerve segments from C57Bl/6J mice (F6P/G6P: fructose-6-phosphate/glucose-6-phosphate. FBP: fructose-1,6-bisphosphate. GI-OH3P: Glyceraldehyde-3-phosphate. 2PG/3PG: 2-phosphoglycerate/3-phosphoglycerate. LACT: lactate) (Error bars represent s.e.m. n=5 mice per condition and metabolite). b, Top: Scheme of extracellular flux analysis of SCs purified from C57Bl/6J mouse nerves. Bottom: Box and whiskers plot (maximum, 25th percentile, median, 75th percentile, minimum) shows glycolytic activity parameters as assessed by extracellular acidification rate (ECAR) measurements in control mouse SCs and cells with Nrg1-induced ErbB2 activation (n=9 well preparations per condition, *P=0.0080, **P=0.0464, ***P<0.0001). c, Western blot analysis (cropped blot images) of control- and Nrg1-treated C57Bl/6J mouse SCs probed with the indicated antibodies. (n=6 independent pair preparations (2 separate dishes) for PFKFB3, and n=3 independent pair preparations (2 separate dishes) for LDHA quantification). d, Intracellular and extracellular (supernatant) lactate concentrations from control and Nrg1-treated mouse SC preparations normalized to cell number and cellular protein. Note decreased intracellular and increased extracellular lactate levels in SCs treated with Nrg1 for 24h, indicating greatly increased lactate extrusion (Error bars represent s.e.m. n=3 well preparations from 3 independent experiments per condition).

Article Snippet: SCs were subsequently control-treated or treated with 200 ng/ml recombinant Nrg1 (R&D Systems, 396-HB-050) for 24h, collected in RIPA buffer containing phosphatase and protease inhibitors, and then processed for protein analysis and western blotting using standard procedures.

Techniques: Control, Purification, Activity Assay, Activation Assay, Western Blot

Journal: eLife

Article Title: Axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons

doi: 10.7554/eLife.104069

Figure Lengend Snippet:

Article Snippet: To investigate the in vitro formation of the stem axon in DRG neuron development, we transduced these cells with an ultra-purified recombinant AAVPHP.S-CMV-eGFP (#VB010000-9394npt, VectorBuilder) virus at DIV 9 at a concentration of 1.12 × 10 10 cfu/ 7000 cells.

Techniques: Knock-Out, Transfection, Construct, Plasmid Preparation, Transduction, IF-cells, Sequencing, RNAscope, Blocking Assay, Multiplex Assay, Software, Staining, Formulation, Immunofluorescence, Amplification, Live Cell Imaging, Imaging, Western Blot

Journal: PLoS ONE

Article Title: Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma

doi: 10.1371/journal.pone.0160933

Figure Lengend Snippet: Differential effects of SMLEF bipolar pulses, SMHEF pulses and thrombin on PRP activation, growth factor release, and cell proliferation.

Article Snippet: Growth factor-dependent cell proliferation was confirmed by addition of purified recombinant human EGF (Lonza, Portsmouth, NH, USA).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma

doi: 10.1371/journal.pone.0160933

Figure Lengend Snippet: PRP, treated as described in the Methods, was centrifuged, the supernatant recovered and assayed for pro- and anti-angiogenic factors by ELISA and for stimulation of cell proliferation using serum-starved epithelial cells (MCF10A). Cell proliferation in response to the plasma supernatants of unactivated or activated PRP (panel E) is normalized to that obtained with supernatants of unactivated PRP. Purified recombinant human EGF 100 ng/mL added to serum-free media increased cell proliferation 1.75-fold relative to media alone (data not shown). Results shown are means ± SEM, n = 5. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Growth factor-dependent cell proliferation was confirmed by addition of purified recombinant human EGF (Lonza, Portsmouth, NH, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Purification, Recombinant

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-c-Myc , Abcam , Cat#Ab32072; [Y69]; RRID: AB_731658.

Techniques: Recombinant, In Vivo, Protease Inhibitor, Plasmid Preparation, Purification, Sample Prep, Sequencing, RNA Sequencing Assay, shRNA, Software

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-C: Bacterially purified GST-Bub2 or GST-Bub2-Q132L, MBP-Bfa1 and 6xHis-Tem1 proteins were used to measure the kinetics of hydrolysis+dissociation (γ[ 32 P]GTP) or dissociation only (γ[ 35 S]GTP) using a filter binding assay (see ). Graphs show average values and standard deviations from three independent experiments. D: Exponentially growing cultures of the indicated strains were shifted to nocodazole containing medium at t = 0. Cell samples were withdrawn at the indicated time for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a checkpoint defect (indicated by re-budding in the absence of proper chromosome segregation) was scored in cycling cultures of the indicated strains shifted either to 14°C for 16h (left graph) or to 37°C for 3h (right graph). F-G: Exponentially growing cells with the indicated genotypes were arrested in G1 by α-factor and released into fresh medium at time 0. At 70’ after release α-factor was re-added to prevent cells from entering a second cell cycle. Cell samples were collected for FACS analysis of DNA contents (F) and for tubulin staining by indirect immunofluorescence (G). H: Cells were treated as in (F-G). TCA extracts were prepared from cell samples at the indicated time points to monitor kinetics of Bfa1-HA6 phosphorylation and Clb2 accumulation and degradation by western blot analysis. Pgk1 was used as loading control. I: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. J-K: Localization of eGFP- tagged Bub2/Bub2-Q132L, Tem1, Bfa1 (J) and Cdc15-GFP (K) was analysed by fluorescence microscopy after formaldehyde fixation.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Purification, Filter-binding Assay, Staining, Immunofluorescence, Phospho-proteomics, Western Blot, Control, Expressing, Immunoprecipitation, Fluorescence, Microscopy

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Cycling cells co-expressing Spc72-Bfa1-eGFPand Spc42-mCherry to mark the SPB (upper panel) or co-expressing Tem1-eGFP and Tub1-GFP (to mark microtubules, lower panel) were analysed to study the distribution of Spc72-Bfa1-eGFP (A) and Tem1-eGFP (B) at SPBs in SPC72-BFA1 bfa1Δ cells. C-D: Cycling cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). Cell samples were withdrawn at the indicated times for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a SPOC defect was scored after propidium iodide staining of cycling cultures of cells with the indicated genotypes after shift to 14°C for 16h. The histograms on the right side represent the DNA contents of the same cells as measured by FACS analysis. F: Percentage of metaphase cells with Cdc15-GFP at 0, 1 or 2 SPBs was scored in the indicated strains after formaldehyde fixation. Metaphases were identified by means of the Tub1-mCherry co-expressed marker. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD and incubated at the indicated temperature. H: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YP medium containing either glucose or galactose and incubated at 25°C for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Expressing, Staining, Marker, Incubation

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A: Bacterially purified 6XHis-Tem1 and 6XHis-Tem1-Q79L were loaded with γ[ 32 P]GTP either in the absence or in the presence of recombinant MBP-Bfa1 and incubated at 30°C for 10 minutes. The mixture was then added to GST-Bub2 or buffer alone and kinetics of GTP hydrolysis and dissociation was followed by a filter-binding assay (see details in ). Graphs show average values and standard deviations from three independent experiments. B: Wild type and TEM1-Q79L cells were arrested in G1 by α-factor and then released into fresh medium at 25°C (t = 0). Cell samples were withdrawn every 10’ to measure kinetics of budding and spindle formation/elongation after in situ immunostaining of tubulin. C: Actomyosin ring contraction has been visualized by live cell imaging of wild type and TEM1-Q79L expressing Myo1-GFP (n = 30). D: Logarithmically growing cultures of cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. E: The percentage of cells with binucleate cell bodies accompanied or not by SPOC defect was scored after DAPI staining of cycling cells of the indicated strains shifted to 14°C for 16h. F: Logarithmically growing cultures of strains with the indicated genotypes were shifted to nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD or YP galactose plates and incubated at 30°C for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Purification, Recombinant, Incubation, Filter-binding Assay, In Situ, Immunostaining, Live Cell Imaging, Expressing, Flow Cytometry, Staining

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: List of non-essential genes implicated in microtubules dynamics or spindle positioning identified in the SGA screen with TEM1-Q79L .

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Migration

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-PK, anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. C-F: Localization of eGFP- tagged Tem1 and Tem1-Q79L (C-D) or Bfa1-eGFP (E-F) was analysed in the indicated strains by fluorescence microscopy after formaldehyde fixation. Metaphase and anaphase cells were identified by means of the Tub1-mCherry co-expressed marker. Micrographs show representative cells of each strain in anaphase. G: Fluorescence intensity ratios were calculated between the two SPBs in anaphase cells of the indicated strains (see details in ).

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Expressing, Immunoprecipitation, Western Blot, Fluorescence, Microscopy, Marker

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Distribution of Cdc15-GFP (A) or Mob1-GFP (B) was analysed in the indicated strains by fluorescence microscopy after formaldehyde fixation. Metaphase and anaphase cells were identified by means of the Tub1-mCherry co-expressed marker. Micrographs show representative wild type and TEM1-Q79L cells expressing Cdc15-GFP in metaphase. C: Serial dilutions of stationary phase cells with the indicated genotypes were spotted on YPD and incubated at the indicated temperatures for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Fluorescence, Microscopy, Marker, Expressing, Incubation