Journal: Neurochemical Research

Article Title: Morin Improves Cognitive Deficits in an in Vivo Model of Vascular Dementia by Modulating the N-methyl-D-aspartate Receptor Signaling Pathways

doi: 10.1007/s11064-026-04717-7

Figure Lengend Snippet: Morin modulated the expression of NMDA receptors in the hippocampus of VaD rats. a the expression levels of NR1 ; b the expression levels of NR2A ; c the expression levels of NR2B ; d the expression levels of NR1 protein; e the expression levels of NR2A protein; f the expression levels of NR2B protein; g protein levels of p-CREB; h protein levels of p-CAMK2A; i protein levels of p-CAMK2D. Protein levels of NR1, NR2A, and NR2B were quantified by ELISA. Data are presented as mean ± SD ( n = 8 per group). Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test (data met assumptions of normality and homoscedasticity)/Kruskal-Wallis with Dunn’s test (data did not meet assumptions). * indicates a significant difference from the Sham group; # indicates a significant difference 2VO group; *# indicates a significant difference from both the Sham and 2VO groups, with a p-value of less than 0.05 considered statistically significant

Article Snippet: Moreover, phosphorylation levels of calcium/calmodulin-dependent protein kinase II isoforms CAMK2A and CAMK2D at Thr286 (p-CAMK2A, p-CAMK2D) were quantified using the Colorimetric Cell-Based ELISA Kit (CAMK2A/CAMK2D (Phospho-Thr286), Boster Bio, #EKC2366).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein binds to and enters the cell through STEAP2 (A) Comparison of the cell-binding capacity of SARS-CoV-2 wild type (WT) N protein and Omicron N protein expressed in either E. coli or mammalian cells. 1 × 10 5 A549 cells were used to mixed with 1 μg WT N or Omicron N proteins. One hour after protein addition, allophycocyanin (APC) conjugated anti-His antibody was used to detect the cell binding capacity of WT N protein or Omicron N protein. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). (B) Antibody blocking assay. Aliquots of 10 μg of SARS-CoV-2 N protein were pre-mixed with 0, 1, 3, 10, 30, and 100 μg of normal mouse IgG or anti-N monoclonal antibody (NP-mAb-40) and incubated at 4°C overnight. The antibody/N protein complex was used for the A549 cell surface binding assay. The blocking capacity of anti-N antibody was normalized to N protein only control. (C) Membrane fractions of A549 and HPAEpiC cells were extracted and incubated with N protein conjugated beads for 3 h binding at 4°C, and pull-downed for LC-MS-MS analysis (upper panels). A549 and HPAEpiC cells were suspended and treated with N protein for 1 h on ice. After incubation, cells were crosslinked with 3 mM DTSSP for 1.5 h. Then, cells were lysed in RIPA lysis buffer, and N protein complex in the lysate was immunoprecipitated for LC-MS-MS analysis (lower panels). Y axis denotes −logP values while the X axis shows log2 fold change values. Orange dots highlight the statistically significant proteins, with p value < 0.05 (-Log p > 1.3) and fold change>2, and the enriched plasma membrane protein was labeled on the plot. Identified proteins were further sorted by HuMemProtDB. (D) To knock-down (KD) STEAP2 expression, HPAEpiC cells were infected with lentivirus carrying STEAP2 shRNA followed by puromycin selection for 14 days. The STEAP2 mRNA expression levels were assessed by qRT-PCR, and the relative KD efficiency of shSTEAP2 was compared to shLacZ control (left-hand side panel). N protein binding capabilities to HPAEpiC STEAP2 KD cells and shLacZ control KD cells were assessed by flow cytometry analysis, and data were shown as mean fluorescence intensity (MFI). (right-hand side panel). (E) Western blot analysis of STEAP2 in wild type (WT) and knock-out (KO) A549 cells were shown. N protein binding to A549 STEAP2 KO cells was assessed by flow cytometry analysis and shown as mean fluorescence intensity (MFI). Ccr (crotonyl-CoAcarboxylase/reductase, a bacterial protein) binding was used as a control. (F) SARS-CoV-2 N protein enters alveolar cells. HPAEpiC cells were treated with 10 μg SARS-CoV-2 N protein overnight and then stained with anti-N antibody. The localization of N protein (Red) was checked by fluorescence microscope and cell morphology was observed by dimensional interference contrast (DIC). Nuclei of cells were stained by DAPI (blue). (G) N protein entering cells by endocytosis and N protein co-localization with STEAP2. HPAEpiC alveolar cells were seeded on 8 well slides. Cells were pretreated with endocytosis inhibitors HCQ, or Dynasore. Then the cells were treated with N protein overnight. After treatment, the cells were stained by specific antibodies to detected N protein (red), endosome marker (EEA1) (green), and STEAP2 (yellow). Cells were observed under fluorescent microscopy (Invitrogen tech.). Scale bar: 50 μm. All data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; t test. See also Figure S5 .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Comparison, Binding Assay, Flow Cytometry, Fluorescence, Antibody Blocking Assay, Incubation, Blocking Assay, Control, Membrane, Liquid Chromatography with Mass Spectroscopy, Lysis, Immunoprecipitation, Clinical Proteomics, Labeling, Knockdown, Expressing, Infection, shRNA, Selection, Quantitative RT-PCR, Protein Binding, Western Blot, Knock-Out, Staining, Microscopy, Marker

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein delivers nucleic acids into cells (A) N protein-RNA complex binding to the cell surface. Aliquots of 10 μg SARS-CoV-2 N protein were incubated with 1 μg of indicated RNAs for 1 h at 4°C, and added to A549 or HPAEpiC cultures. SARS-CoV-2 N protein only without RNA was used as a control. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). Data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; t test. (B) The observation of N protein-RNA enters into cells. HPAEpiC were seeded onto 8-well glass slides (40,000 cells/well). SARS-CoV-2 N protein 10 μg and 40 μg RNA-FAM (green) were mixed for 1 h at 4°C. cells were treated with SARS-CoV-2 N-RNA-FAM mixture for 1 h. The groups of non-treated cells and RNA-FAM only were as controls. After treatment, N protein was detected by anti-N antibody (Red). The localization of RNA-FAM was green. DAPI (blue) indicates cell nuclei. Scale bar: 15 μm. (C) Lattice light sheet microscopy time lapse imaging of N protein-RNA complex entering into HPAEpiC cells. SARS-CoV-2 N protein 10 μg was mixed with 40 μg RNA-FAM (fluorescein) for 1 h at 4°C and then treated with ice-cooled alveolar cells. The signals of RNA-FAM and Hochest 33,342 were monitored by lattice light sheet microscopy at different time points.

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Binding Assay, Incubation, Control, Flow Cytometry, Fluorescence, Microscopy, Imaging

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein-assisted nucleic acid dispersion and expression in the co-culture environment (A)The co-culture system consisted of A549 as recipient cells, and 293T pre-transfected with two plasmids, one expressing GFP and the other expressing SARS-CoV-2 N protein or the pcDNA3.1 empty vector. (B–E) After 24 h co-culture of the donor cells and recipient cells, cell pool was stained with cytokeratin 18 (an A549 marker) and SV40 large T antigen (a 293T marker). A549 cells in the cell pool were gated from cytokeratin 18 positive and large T antigen negative. A549 GFP positive percentage was further assessed by flow cytometry analysis. Effects of SARS-CoV-2 N variants (B), treatment with RANTES (C), the p38 inhibitor SB203580 (D), or anti-N neutralizing antibody (E) were accessed by adding these effectors to the medium. Experiments are performed in three to five biological replicates. ∗, p value <0.05 (paired two-tailed student’s t -test). (F) SARS-CoV-2 N protein promotes gene delivery by cell-free diffusion to neighboring cells. A549 cells were plated in the lower chamber, while 293T donor cells co-transfected with plasmids expressing EGFP and indicated N proteins in the upper chamber. After 3 days of co-culture, GFP positive A549 cells were observed and counted. See also Figures S10 and .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Dispersion, Expressing, Co-Culture Assay, Transfection, Plasmid Preparation, Staining, Marker, Flow Cytometry, Two Tailed Test, Diffusion-based Assay

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet:

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Bioprocessing, Recombinant, Magnetic Beads, Protease Inhibitor, Sequencing, Modification, SYBR Green Assay, shRNA

Journal: Journal of the National Cancer Institute

Article Title: Use of three-dimensional tissue cultures to model extravascular transport and predict in vivo activity of hypoxia-targeted anticancer drugs.

doi: 10.1093/jnci/djj306

Figure Lengend Snippet: Fig. 1. HT29 multicellular layer (MCL) and diffusion chamber apparatus. Transverse hematoxylin and eosin-stained section ( right ) of an HT29 MCL and a schematic depiction of its use in a diffusion chamber to determine extravascular transport properties ( left ). Drug is introduced to the donor (D) compartment, and then samples are taken from both the donor and receiver (R) compartments at intervals for analysis.

Article Snippet: HT29 human colon carcinoma cells from American Type Culture Collection (Manassas, VA) were cultured as monolayers in α minimal essential medium ( α MEM, Gibco, Invitrogen Corporation, Carlsbad, CA ) with 5% heat-inactivated fetal bovine serum (FBS, Gibco).

Techniques: Diffusion-based Assay, Staining

Journal: Journal of the National Cancer Institute

Article Title: Use of three-dimensional tissue cultures to model extravascular transport and predict in vivo activity of hypoxia-targeted anticancer drugs.

doi: 10.1093/jnci/djj306

Figure Lengend Snippet: Fig. 3. Predictions of drug exposure and cell killing in the modeled tumor microvascular network. The microvascular network depicted in Fig. 2, A was used for this analysis. A ) Predicted tirapazamine (TPZ) pharmacokinetics in tumor tissue relative to that in plasma, using parameters measured in HT29 tumor cells. Each point represents the area under the concentration – time curve (AUC) calculated by the transport model plotted against oxygen concentration calculated from the same model at that particular position in the tumor microregion. Points were randomly selected in the three-dimensional tumor microregion. Drug exposure of hypoxic cells is lowered due to metabolic depletion of drug as it diffuses from the vasculature. B ) Predicted TPZ pharmacodynamics. Fraction of tumor cells surviving at each position in the tumor microregion, as calculated by the three-dimensional pharmacokinetic/pharmacodynamic model (based on clonogenic assays), was plotted against calculated oxygen concentration at the same position. Solid curve shows the predicted survival when TPZ concentration equals plasma concentration. The difference between points and curve shows compromised cell killing resulting from the limitation to extravascular transport.

Article Snippet: HT29 human colon carcinoma cells from American Type Culture Collection (Manassas, VA) were cultured as monolayers in α minimal essential medium ( α MEM, Gibco, Invitrogen Corporation, Carlsbad, CA ) with 5% heat-inactivated fetal bovine serum (FBS, Gibco).

Techniques: Drug discovery, Clinical Proteomics, Concentration Assay

Journal: Journal of the National Cancer Institute

Article Title: Use of three-dimensional tissue cultures to model extravascular transport and predict in vivo activity of hypoxia-targeted anticancer drugs.

doi: 10.1093/jnci/djj306

Figure Lengend Snippet: Fig. 4. Determination of the parameters of the spatially resolved pharmacokinetic/pharmacodynamic model for tirapazamine (TPZ) analogs. Representative data are shown for several compounds: TPZ, ○ ; 1 , □ ; 2 , + ; 5 , Δ ; 6 , ; 7 , ␣ ; 9 , ◊ ; 13 , ×. The resulting parameter estimates as shown in Table 1 . A ) Concentrations in the receiver compartment of the diffusion chamber, as a fraction of initial value in the donor compartment ( C 0 ). Mean HT29 multicellular layer thicknesses determined from 14 C-urea diffusion were (in μ m): TPZ, 176; 1 , 169; 2 , 177; 5 , 132; 6 , 144; 7 , 137; 9 , 181; and 13, 164. Curves are fi ts to a diffusion model, from which the diffusion coeffi cient ( D ) of each drug was estimated. B ) Extracellular drug concentration as a fraction of the initial value in anoxic stirred HT29 cell suspensions, which were used to determine the fi rst-order rate constant for drug metabolism ( k met,0 ). C ) Cell survival in the same experiments plotted against the drug exposure integral, E (the integrated from of Equation 1c , see Materials and Methods). The gradient, γ , is the proportionality constant relating log cell survival to E. D ) Evaluation of plasma pharmacokinetics of TPZ and analogs at their maximum tolerated doses. Total drug in plasma was measured by high-performance liquid chromatography. Values are mean with upper or upper and lower 95% confi dence intervals for three or four mice.

Article Snippet: HT29 human colon carcinoma cells from American Type Culture Collection (Manassas, VA) were cultured as monolayers in α minimal essential medium ( α MEM, Gibco, Invitrogen Corporation, Carlsbad, CA ) with 5% heat-inactivated fetal bovine serum (FBS, Gibco).

Techniques: Diffusion-based Assay, Concentration Assay, Clinical Proteomics, Drug discovery, High Performance Liquid Chromatography

Journal: Journal of the National Cancer Institute

Article Title: Use of three-dimensional tissue cultures to model extravascular transport and predict in vivo activity of hypoxia-targeted anticancer drugs.

doi: 10.1093/jnci/djj306

Figure Lengend Snippet: Fig. 6. Comparison of measured and model-predicted killing of hypoxic cells in HT29 tumors. Gain in logs of cell kill over radiation alone was measured by determining the surviving fraction of cells in tumors excised 18 hours after treatment of mice with 20 Gy radiation alone and in combination with the maximum tolerated dose of each compound. Values are mean ± 95% confi dence interval for three to fi ve tumors. A ) Prediction based on the spatially resolved pharmacokinetic/pharmacodynamic model. B ) Prediction using the same oxygen distribution but assuming that the pharmacokinetics throughout the tumor is the same as in plasma (i.e., infi nitely rapid extravascular transport). Open symbols , compounds that are inactive in tumors (i.e., no statistically signifi cant increase in log cell killing compared with radiation alone); solid symbols , compounds that are active by this defi nition.

Article Snippet: HT29 human colon carcinoma cells from American Type Culture Collection (Manassas, VA) were cultured as monolayers in α minimal essential medium ( α MEM, Gibco, Invitrogen Corporation, Carlsbad, CA ) with 5% heat-inactivated fetal bovine serum (FBS, Gibco).

Techniques: Comparison, Drug discovery, Clinical Proteomics

Journal: Journal of cell science

Article Title: CCM1 regulates vascular-lumen organization by inducing endothelial polarity.

doi: 10.1242/jcs.059329

Figure Lengend Snippet: Fig. 4. CCM1 is required for junctional localization of the components of the polarity complex Tiam, Par3 and phosphorylated PKCz in cultured ECs and in human CCM1 vascular lesion. (A,B) Junctional staining of Tiam, Par3 and phosphorylated PKCz, in both Ccm1-silenced VEC-positive cells and in Ccm1 fl/fl+Cre cells, appears convoluted and fragmented (arrowheads). This pattern is reminiscent of VEC and b-catenin distribution in Ccm1-silenced ECs (Fig. 3D,E). Scale bar: 20 mm. (C) Phosphorylation of PKCz at Thr410 (p-PKCz) is downregulated by 40% and 80%, respectively, in CDH5-silenced HUVECs and in VEC-null cells. Phosphorylation of PKCz is not modified after CCM1 or Ccm1 siRNA treatment. Data shown are representative of three independent experiments. Vertical bars indicate that the bands, from the same blot, do not come from contiguous lanes. (D) Brain vascular lesion of a patient with heterozygous mutation of CCM1 gene. Phosphorylated PKCz expression is diffuse over large areas in the endothelium of the vascular mulberry lesion (arrowheads, magnification of boxed area). It is instead concentrated to interendothelial junctions in peri-lesion vessels (arrowheads, magnification of boxed area). Scale bars: 50 mm and 10 mm (in magnifications). Asterisks indicate the nuclei of ECs lining the lumen (dashed line).

Article Snippet: For immunofluorescence (IF), western blot (WB) and immunoprecipitation, the following antibodies were used: VE-cadherin (C-19, sc-6458, goat, Santa Cruz), bcatenin (mouse, BD Transduction Laboratories), CCM1 (residues 1-207 of the human sequence and crossreacting with mouse protein, rabbit, gifts from Elisabeth TournierLasserve, Université de Médicine Paris Diderot, Paris, France and Francesco Retta, University of Torino, Torino, Italy) and (KRIT1, K-16, sc-23997, goat, Santa Cruz), phosphorylated PKCz (rabbit, Cell Signaling), total a-PKCz (C-20, sc-216, rabbit, Santa Cruz), Rap1 (121, sc-65, rabbit, Santa Cruz, for IF), Rap1b (rabbit, Cell Signaling, for WB), Tiam 1 (C-16, sc-872, rabbit, Santa Cruz), Podxl (murine-specific and human-specific, R&D), Coll IV (murine-specific and human-specific, AbD Serotec), Par3 (rabbit, Millipore), Pecam (clone 13.3, rat anti-mouse), Flag, (M2, mouse, Sigma), rac (mouse, BD Transduction Laboratories), cdc42 (rabbit, Cell Signaling), HA (clone 11, mouse, Covance).

Techniques: Cell Culture, Staining, Phospho-proteomics, Modification, Mutagenesis, Expressing

Journal: Journal of cell science

Article Title: CCM1 regulates vascular-lumen organization by inducing endothelial polarity.

doi: 10.1242/jcs.059329

Figure Lengend Snippet: Fig. 5. Rap1b contributes to the polarizing activity of VEC and CCM1. (A) Rap1b regulates the junctional localization of phosphorylated PKCz. In RAP1B-silenced HUVECs, phosphorylated PKCz loses its linear junctional expression pattern (arrowheads), which instead becomes fragmented (arrows). Scale bar: 20 mm. (B) Rap1b requires VEC expression for its activation. Pull-down of GTP-Rap1was followed by western blotting for Rap1b. Two additional experiments gave comparable results. (C) RAP1B-silenced HUVECs show abnormal lumen (L) maturation (24 hours). Podxl and Coll IV apical or basal localization, respectively, is lost (arrowheads and arrows). The dashed lines outline the lumen. Scale bars: 50 mm and 10 mm (magnifications). (D) Rap1 disappears from cell-cell contacts in Ccm1 silenced or Ccm1 fl/fl+Cre cells and often concentrates at focal contacts (arrows and arrowheads). (E) Active Rap1 (HA-Rap1 GV12) concentrates to cell-cell junctions with VEC, in control cells (Ccm1 fl/fl) (arrows). The junctional localization is strongly reduced or lost (arrows) in parallel with diffusion of VEC in Ccm1-silenced (Ccm1 fl/fl+Cre) ECs. Immunofluorescence staining with an antibody against the HA tag present in Rap1 GV12. Scale bar: 20 mm (D,E).

Article Snippet: For immunofluorescence (IF), western blot (WB) and immunoprecipitation, the following antibodies were used: VE-cadherin (C-19, sc-6458, goat, Santa Cruz), bcatenin (mouse, BD Transduction Laboratories), CCM1 (residues 1-207 of the human sequence and crossreacting with mouse protein, rabbit, gifts from Elisabeth TournierLasserve, Université de Médicine Paris Diderot, Paris, France and Francesco Retta, University of Torino, Torino, Italy) and (KRIT1, K-16, sc-23997, goat, Santa Cruz), phosphorylated PKCz (rabbit, Cell Signaling), total a-PKCz (C-20, sc-216, rabbit, Santa Cruz), Rap1 (121, sc-65, rabbit, Santa Cruz, for IF), Rap1b (rabbit, Cell Signaling, for WB), Tiam 1 (C-16, sc-872, rabbit, Santa Cruz), Podxl (murine-specific and human-specific, R&D), Coll IV (murine-specific and human-specific, AbD Serotec), Par3 (rabbit, Millipore), Pecam (clone 13.3, rat anti-mouse), Flag, (M2, mouse, Sigma), rac (mouse, BD Transduction Laboratories), cdc42 (rabbit, Cell Signaling), HA (clone 11, mouse, Covance).

Techniques: Activity Assay, Expressing, Activation Assay, Western Blot, Control, Diffusion-based Assay, Immunofluorescence, Staining

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Single-Cell Survey Reveals Heterogeneity of cDC2s with Two Subsets Delineated by Expression of T-Bet (A) Representative contour plot showing gating strategy for splenic DCs in Tbx21 RFP-Cre mice. DCs defined as Lin(CD3,CD19,CD49b,Siglec-F) – Ly6C – CD64 – CD11c + MHCII + . (B) Frequency of T-bet + cDC2s across tissues. Each circle represents one mouse. In the peripheral and mesenteric LN (PLN and MLN), migratory DCs were defined as MHCII hi CD11c int and resident DCs as MHCII int CD11c hi . Error bars represent mean ± SEM. (C) Analysis of RFP + and YFP + splenic cDC2s from Tbx21 RFP-CreERT2 Rosa26 YFP mice, 3 days post tamoxifen gavage. (D) Percent RFP + and YFP + of cDC2 cells. Percent RFP + of YFP + cDC2s at indicated time points post tamoxifen gavage (right). Error bars represent mean ± SEM; n = 3–4 mice per time point. (E) t-SNE embedding of 4,464 DCs. Colors indicate unsupervised clustering by Phenograph (left panel) or classification based on expression of canonical markers (right panel). (F) Expression of canonical DC markers across the transcriptionally defined DC clusters from (E). (G) Proportion of T-bet (RFP + ) cells in each cell cluster identified in (D). (H) Violin plot showing expression of the cell-cycle signature across the DC clusters from (E). (I) Similarity of bulk T-bet – cDC2s, T-bet + cDC2, and cDC1 transcriptomes to the reference single-cell DC clusters (E). Colors represent the correlation coefficient between the cell population identified in the row label and the DC cluster identified by the column label. See also and .

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Single-Cell Survey Reveals Heterogeneity of cDC2s, Related to Figure 1 A. Representative histogram showing expression of T-bet (RFP) in splenic cells from Tbx21 RFP-cre mice. (B). Expression of T-bet in CD11b + XCR1 + DCs from the intestinal lamina propria. Data representative of > 5 independent experiments, with at least 3 mice per experiment. (C). Expression of T-bet in splenic myeloid cells. Cells were defined as: (i) Ly-6C hi monocytes (Lin – Ly6C + Ly6G – CD11b + CX3CR1 + ); neutrophils (Lin – Ly6C + Ly6G + ); macrophages (Lin – CD64 + Ly6C – ). Lineages (Lin) were defined as: CD3e, CD90.2, CD19, CD49b and Siglec F. Each circle represents an individual mouse, error bars represent mean ± SEM. (D). Left: Gating strategy for single-cell sorting. DCs were defined as Lin(CD3, CD19, CD90) – Ly6C – CD64 – CD11c + MHCII + . Two populations were sampled: RFP + DCs and RFP – DCs (encompassing XCR1 + cDC1s, CD11b + RFP – and CD11b – XCR1 – DCs). Right: Post-sort purity of RFP + and RFP – cells. Contaminating population of Ly6C + cells identifiable on post-sort purity (lower panel). (E). Similarity of splenic CD11c + MHCII + cells to reference myeloid cells (ImmGen Consortium) Colors represent the Pearson correlation between the mean gene expression from the dendritic cell cluster in the rows and the bulk reference transcriptome in the columns. (F). Top 20 positive and negative gene loadings of PC1 for T-bet + cDC2 clusters after cell-cycle correction (left panel). Scatterplot of PC1 and PC2 for T-bet + cDC2 clusters after cell-cycle correction (right panel).

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing, FACS, Gene Expression

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Environmental Cues Drive Distinct DC2 Differentiation Pathways within the Spleen, Related to Figure 5 (A). Gating strategy for the identification of DC progenitors in the bone marrow (BM) (B). Palantir pseudo-time analysis of differentiation potential and branch probabilities from the Siglec-H + pre-DC state to T-bet + cDC2 and T-bet – cDC2 terminal states. (C). Plots showing Palantir differentiation potential (y axis) along Palantir pseudo-time (x axis) for Siglec-H + DC and T-bet + cDC2s (top) or Siglec-H + DC and T-bet – cDC2 clusters (bottom) (D). Plots showing the top two diffusion component embeddings for Siglec-H + DC and T-bet + cDC2 clusters (top) or Siglec-H + DC and Tbet – cDC2 clusters (bottom). Black arrow indicates Siglec-H + DC cluster cells adjacent to cells from the proliferative T-bet + cDC2 clusters 6 and 8. (E). Top panel: plots showing probability of each cell being within 20 nearest neighbors of randomly sampled shortest paths from the Siglec-H + DC to the indicated end points. Middle panel: plots showing the proportion of cells belonging to Siglec-H + DC, T-bet + cDC2, or T-bet – cDC2 from 20 nearest neighbors of randomly sampled shortest paths. Bottom: plots showing diffusion distance step sizes for each step along the indicated shortest paths (bottom panel). Colors illustrate cluster membership. (F). Graph showing AUC (x axis) for genes differentially expressed between Siglec-H + DC cluster (cluster 11) and all other cDC2 clusters. EMD on the y axis. Dashed lines represents μ EMD ± 3σ EMD . (G). Gating strategy for FACS-isolation of MHCII + ILC3s: Lin = CD3, CD19, CD49b, Siglec-F. (H). Heatmap reports scaled expression of 3550 differentially expressed genes (log 2 FC > 1, FDR < 0.01) between ILC3s and Rorγt fm cDC2s. Selected genes listed to the right. (I). Representative flow cytometric analysis of phenotypes of splenic progeny from Tbx21 RFP-cre CD45.2 + Ly6C − CD64 – MHCII + CD11c + Siglec-H + pre-DCs adoptively transferred into sub-lethally irradiated CD45.1 recipient mice 7 days earlier (data from one experiment with n = 3). J. Sort purified T-bet + or T-bet – cDC2 were cultured for 24hrs in the presence of LPS, CpG, TNF-α or IFN−γ. Representative overlay histogram showing the expression of RFP(T-bet) at 24hrs. Data representative of 2 (TNF-α) or 4 (all other cytokines/TLR agonists) independent experiments, n = 2-3.

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Diffusion-based Assay, Isolation, Expressing, Irradiation, Purification, Cell Culture

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Human DC Heterogeneity, Related to Figure 7 (A). Violin plots showing expression distribution of mouse DC subset marker genes across human peripheral blood DC and monocyte clusters identified in Villani et al. (2017) . (B). Representative flow cytometric analysis of mouse peripheral blood cDC2s showing absence of T-bet (RFP) + cDC2s. (C). Gating strategy for FACS-isolation of human spleen DCs for scRNA-seq. DCs were defined as live, LIN(CD3,CD56,CD19) − CD14 – CD11C + HLA-DR + . (D). Representative flow cytometry analysis of human spleen cDC2s gated as Lin(CD3,CD56,CD19) – CD14 – CD11c + HLA-DR + CD123 – XCR1 – CLEC4A + cells. Left panel: cell surface expression of CD1c and CLEC10A by cDC2s. Right panel: overlay of CLEC10A + and CLEC10A – cDC2s distinguished by differential expression of CLEC4A and FcεR1a. Summary bar graphs show frequency of CD1C + CLEC10A + and CD1C + CLEC10A – cDC2s as a percentage of cDC2s (n = 4 individuals). (E). t -SNE embedding of 9,315 FACS-isolated CD45 + immune cells from two melanoma tumors. Colors indicate unsupervised clustering by Phenograph (left panel) or classification based on expression of canonical markers and correlations with bulk RNA-seq data (right panel). Each dot represents an individual cell. (F). Pearson correlations between cluster centroids in (F) and bulk RNA-seq data from purified immune populations ( Jeffrey et al., 2006 , Novershtern et al., 2011 ) (G). t-SNE map of 2,122 myeloid cells identified in (F). Colors indicate patient sample (left) or unsupervised clustering by Phenograph (right panel). Each dot represents an individual cell. (H). Heatmap of normalized, log transformed and MAGIC imputed expression of top 20 differentially expressed genes, defined by the highest earth mover’s distance (EMD), per Phenograph cluster in E. The colored bar at the top of the heatmap shows assignment of cells to clusters labeled in F, right panel. (I). t-SNE map of human melanoma myeloid cells (H) colored by imputed expression of labeled genes.

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing, Marker, Isolation, Flow Cytometry, Quantitative Proteomics, RNA Sequencing, Purification, Transformation Assay, Labeling

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Recombinant, Staining, Multiplex Assay, Cell Isolation, Gene Expression, Software

Journal: Virchows Archiv

Article Title: Testing for ROS1 in non-small cell lung cancer: a review with recommendations

doi: 10.1007/s00428-016-2000-3

Figure Lengend Snippet: Prevalence of ROS1 rearrangements in non-small cell lung cancer screening studies (modified from Gainor and Shaw 2013 [ 35 ] a )

Article Snippet: These results are based on the use of the ROS1 (D4D6) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) applied at dilutions ranging from 1:50 to 1:1000 with various antigen retrieval methods and use of different amplification and detection systems, in automated instruments or manually.

Techniques: Modification, Sequencing, Phospho-proteomics, DNA Sequencing

Journal: Virchows Archiv

Article Title: Testing for ROS1 in non-small cell lung cancer: a review with recommendations

doi: 10.1007/s00428-016-2000-3

Figure Lengend Snippet: Summary of the clinical studies of crizotinib in ROS1 -positive NSCLC

Article Snippet: These results are based on the use of the ROS1 (D4D6) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) applied at dilutions ranging from 1:50 to 1:1000 with various antigen retrieval methods and use of different amplification and detection systems, in automated instruments or manually.

Techniques:

Journal: Virchows Archiv

Article Title: Testing for ROS1 in non-small cell lung cancer: a review with recommendations

doi: 10.1007/s00428-016-2000-3

Figure Lengend Snippet: Examples of different FISH signal patterns using ROS1 break-apart assays. a–d Vysis LSI ROS1 (Cen) SpectrumGreen Probe and Vysis LSI ROS1 (Tel) SpectrumOrange Probe (Abbott Molecular, IL, USA) on histological specimens. a Normal (negative) ROS1 pattern: two fused signals. b Typical ROS1 -positive pattern with fused and split signals. c Atypical ROS1 -positive pattern with one fusion signal and isolated 3′ green signals. d Increased ROS1 copy number. This pattern should not be interpreted as positive; e–f ZytoLight SPEC ROS1 (Cen) Green Probe and (Tel) Orange Probe (ZytoVision, Bremerhaven, Germany) on cytological specimens. e Split signals. f Isolated 3′ green signals

Article Snippet: These results are based on the use of the ROS1 (D4D6) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) applied at dilutions ranging from 1:50 to 1:1000 with various antigen retrieval methods and use of different amplification and detection systems, in automated instruments or manually.

Techniques: Isolation

Journal: Virchows Archiv

Article Title: Testing for ROS1 in non-small cell lung cancer: a review with recommendations

doi: 10.1007/s00428-016-2000-3

Figure Lengend Snippet: Criteria for dual-colour break-apart FISH detection of ROS1 rearrangements in NSCLC

Article Snippet: These results are based on the use of the ROS1 (D4D6) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) applied at dilutions ranging from 1:50 to 1:1000 with various antigen retrieval methods and use of different amplification and detection systems, in automated instruments or manually.

Techniques: Isolation, Cell Counting

Journal: Virchows Archiv

Article Title: Testing for ROS1 in non-small cell lung cancer: a review with recommendations

doi: 10.1007/s00428-016-2000-3

Figure Lengend Snippet: a – f Examples of ROS1 IHC in histological NSCLC specimens (D4D6 antibody, Ventana BenchMark XT; DAB chromogen). a HCC78 cell line (cellblock; ×400). b NSCLC with diffuse, strongly positive staining (×200). c NSCLC with diffuse, granular cytoplasmic staining (×400). d Adenocarcinoma with heterogeneous staining (×200). e Non-neoplastic type II pneumocytes with weak ROS1 staining (×630). f Bone metastasis of a ROS1 -negative NSCLC showing strong granular staining of non-neoplastic osteoclastic giant cells (×400). g–h Aberrant immunostaining of ROS1 in a transbronchial biopsy with lung adenocarcinoma. g H&E stain, asterisks show tumour cells. h ROS1 IHC in adjacent hyperplastic type II pneumocytes ( arrows ) but not in tumour cells ( asterisks )

Article Snippet: These results are based on the use of the ROS1 (D4D6) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) applied at dilutions ranging from 1:50 to 1:1000 with various antigen retrieval methods and use of different amplification and detection systems, in automated instruments or manually.

Techniques: Staining, Immunostaining

Journal: Virchows Archiv

Article Title: Testing for ROS1 in non-small cell lung cancer: a review with recommendations

doi: 10.1007/s00428-016-2000-3

Figure Lengend Snippet: ROS1 IHC in ethanol-fixed and previously Papanicolaou-stained cytological specimens (D4D6 antibody, Leica BondMax; AEC chromogen, ×400). a HCC78 cell line (positive control; cytospin). b ROS1 -positive adenocarcinoma. c Small group of ROS1 -positive adenocarcinoma cells surrounded by numerous benign respiratory epithelial cells

Article Snippet: These results are based on the use of the ROS1 (D4D6) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) applied at dilutions ranging from 1:50 to 1:1000 with various antigen retrieval methods and use of different amplification and detection systems, in automated instruments or manually.

Techniques: Staining, Positive Control

Journal: Virchows Archiv

Article Title: Testing for ROS1 in non-small cell lung cancer: a review with recommendations

doi: 10.1007/s00428-016-2000-3

Figure Lengend Snippet: Commercially available assays for ROS1 testing

Article Snippet: These results are based on the use of the ROS1 (D4D6) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) applied at dilutions ranging from 1:50 to 1:1000 with various antigen retrieval methods and use of different amplification and detection systems, in automated instruments or manually.

Techniques:

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Representative anti-pY Western (upper panel) and far-Western blots (next four panels) of 60-min EGF stimulation time-course. Far-Westerns using GRB2, SHP2-N, NCK1 and RASGAP-N are shown to illustrate major binding patterns identified (see B ). Additional SH2 blot data are provided in . Immunoblotting with antibodies to EGFR and tubulin was used to confirm equal loading. ( B ) Hierarchical clustering of SH2 domains on the basis of binding to four major phosphoproteins (EGFR, GAB1, p130CAS, and SHCA). Signal was normalized to maximum band intensity across all time points and all bands for each probe replicate. Then data for each phosphoprotein was averaged in a probe specific manner (red represents greater percentage of total signal, max = 1, min = 0). Names of SH2/PTB domain probes are indicated on the right. Colored boxes represent SH2 clusters defined by un-centered correlation coefficient >0.85. DOI: http://dx.doi.org/10.7554/eLife.11835.004

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Western Blot, Binding Assay

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) EGFR, ( B ) p130CAS, ( C ) GAB1, and DOI: http://dx.doi.org/10.7554/eLife.11835.006

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques:

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Hierarchical clustering of relative SH2 specificity. Values were obtained by subtracting percent bound by pY from percent bound by each SH2 for each time point. Red represents binding greater than pY and green represents binding less than pY. Relative SH2 specificity value = %Band intensity SH2 at time x - %Band intensity anti-pY at time x. ( B ) Hierarchical clustering of SH2 binding to EGFR band (normalized to maximum, max=1, min=0) for 60-min time-course. ( C ) Hierarchical clustering of EGFR phosphosite-specific Western blotting quantifications (normalized to maximum, max=1, min=0) for 60-min time-course. ( D ) and ( E ) Plots comparing the EGFR phosphosite kinetics and SH2 binding kinetics for SH2 domains and their canonical binding motifs ( D , CRKL and pY992, R 2 =0.81; E, GRB2 and pY1168, R 2 =0.91). ( F ) Plot comparing total EGFR phosphorylation (pY EGFR band) with SHCA PTB binding (R 2 =0.89). R-squared values were calculated by plotting normalized pY Western intensity against normalized SH2 binding FW intensity. pY EGFR quantifications consist of data from a single experiment.( G ) pERK1 and pERK2 pY/pT activation site phosphorylation kinetics following stimulation with EGF. Lines represent average of two technical replicates. DOI: http://dx.doi.org/10.7554/eLife.11835.007

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Binding Assay, Phospho-proteomics, Western Blot, Activation Assay

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: Eight time points; 0, 0.167, 0.5, 1, 1.5, 3, 10 and 30 min post-EGF were analyzed. Immunoblotting with antibodies to EGFR and tubulin was used to confirm equal loading. DOI: http://dx.doi.org/10.7554/eLife.11835.009

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Western Blot

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Venn diagram showing overlap of significant gene ontologies for proteins containing peptides whose phosphorylation was enhanced or unchanged by EGF (p < 0.05, Bonferroni corrected). The number of unique or overlapping ontologies observed for each protein set is indicated within the diagram. GO terms listed represent the three largest GO parent terms returned by REVIGO . ( B ) Amino acid frequency logos for sites whose phosphorylation was enhanced (upper) and unchanged (lower) by EGF stimulation. Background data is PhosphoSitePlus pY database. ( C–F ) Relative phosphopeptide abundance for peptides derived from EGFR ( C ), GAB1 ( D ), SHCA ( E ) and p130CAS (BCAR1) ( F ). Specific phosphopeptide sequences are listed. Results are average of three biological replicates. DOI: http://dx.doi.org/10.7554/eLife.11835.008

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Phospho-proteomics, Derivative Assay

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A-C ) TIRF images of additional fluorescently tagged SH2 domains before and after EGF stimulation. A) GAB1 binding domains (SHP2-NC) ( B ) EGFR binding domains (GRB7) and ( C ) p130CAS binding domains (CRK, RASGAP-NC). Domains are labeled according to clustering results from . Post-EGF images were taken ~40min after stimulation. Scale bars = 10 μm ( D ) Correlation plot of SH2 domain probe diffusion rate ( D , y-axis) and recruitment time constant (τ, x-axis). Data for SHP2-C was an outlier and was removed from the plot for clarity. Error bars for τ values represent SEM (see ). DOI: http://dx.doi.org/10.7554/eLife.11835.011

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Binding Assay, Labeling, Diffusion-based Assay

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Representative white light ( top panel ) and epifluorescence images ( lower panel ) of GRB2 SH2-tdEOS transfected A431 cells that were used to determine total cell number, transfection efficiency and relative expression level. Insert ( upper panel) shows representative DIC image of nonadherent cells used to determine cell volume. ( B ) Histogram of individual cell GRB2 SH2-tdEOS expression levels. Left skew in expression was compensated for in the final calculation. ( C ) Anti-GRB2 SH2 blot used to calculate the average concentration of GRB2 SH2-tdEOS (6.5 μM) and endogenous GRB2 (1.5 μM). Concentrations were determined by using bacterially produced GST-GRB2 SH2 fusion as standard (right side of the blot). ( D ) Anti-pY blot showing EGF-induced EGFR phosphorylation and phosphorylation standard titration used to calculate the cellular concentration of phosphorylated EGFR sites. Concentrations were determined using a highly phosphorylated recombinant ABL standard with a known pY concentration (right side of the blot). ( E ) Representative z-axis cross-sections of fixed A431 cells immunostained with anti-pY. The images and traces were obtained from the same cell along the x- and y-axes. White block indicates the quantified area. Curves represent an average of multiple line scan quantifications across an individual cell membrane. ( F ) Apical and basal pY levels following EGF stimulation as measured by immunofluorescence. Intensity measurements were averaged from two independent experiments; a total of at least seven cells were quantified for each time point. Error is SEM for all data points. ( G ) Ratio of apical to basal phosphorylation following stimulation with EGF. DOI: http://dx.doi.org/10.7554/eLife.11835.013

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Transfection, Expressing, Concentration Assay, Produced, Phospho-proteomics, Titration, Recombinant, Blocking Assay, Membrane, Immunofluorescence

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: Upper left: three cell lines expressing wt EGFR (A431; Cos1 African green monkey kidney fibroblast cells; H226 human lung squamous cell carcinoma cells) were stimulated with 1 ng/ml EGF for 10 min. Expression and tyrosine phosphorylation of EGFR were determined by immunoblotting with anti-EGFR and anti-pY antibodies. Upper right and lower panels: proliferation assay results for A431, Cos1, and H226 cells. Cells in 96-well plates were incubated with control (0.1% FBS), 0.1–25 ng/ml EGF, or 10% FBS medium up to 72 hr as indicated in the inset. Cell proliferation was determined with CYQUANT direct reagents as described in Methods. Fold changes in normalized fluorescence values at 0 hr, 24 hr, and 72 hr after treatment are shown. Asterisks indicate a significant difference between treatments at 72 hr (p<0.0001, one-way ANOVA). Note that in A431 cells, 1 ng/ml EGF is mitogenic while 25 ng/ml EGF suppresses growth/survival. EGF is not mitogenic in Cos1 or H226 cells under conditions used here. DOI: http://dx.doi.org/10.7554/eLife.11835.015

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Expressing, Phospho-proteomics, Western Blot, Proliferation Assay, Incubation, Control, CyQUANT Assay, Fluorescence

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: ( A ) Representative anti-pY immunoblot (upper panel) and Grb2 SH2 far-western blot (lower panel) of A431 cells stimulated with 1 ng/mL EGF and flash frozen at 22 discrete time points. ( B ) Quantification of EGFR tyrosine phosphorylation kinetics (from anti-pY immunoblot) in A431 cells treated with 1 ng/mL EGF (n=2 biological replicates) and 25 ng/mL EGF (n=3 biological replicates). ( C ) Anti-pY immunoblot of A431 cells stimulated with 1 ng/mL and 25 ng/mL EGF at 0, 1.5 and 10 min. EGFR phosphorylation was 5.4 +/- 0.4 fold greater in cells stimulated with 25 ng/mL (normalized for EGFR expression, error = SEM). No difference was observed in prestimulation EGFR phosphorylation after normalization. ( D ) Comparison of Grb2 SH2 binding site phosphorylation kinetics (GST-Grb2 SH2 FW, black; time constant τ = 54.6 +/- 1.4 s, n=2 biological replicates) and Grb2 SH2 in vivo membrane recruitment kinetics (tdEOS-GRB2 SH2 TIRF, red; time constant τ = 116.7 +/- 2.3 s, n=2 biological replicates). DOI: http://dx.doi.org/10.7554/eLife.11835.016

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Western Blot, Far Western Blot, Phospho-proteomics, Expressing, Comparison, Binding Assay, In Vivo, Membrane

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: pervanadate (PV) treated cells. Representative GRB2 SH2 far-Western and anti-pY (PY100) immunoblots for EGF and PV treated cells. Immunoblotting with antibodies to EGFR and actin was used to confirm equal loading. DOI: http://dx.doi.org/10.7554/eLife.11835.022

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Western Blot

Journal: eLife

Article Title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

doi: 10.7554/eLife.11835

Figure Lengend Snippet: CRK SH2 shows little or no binding to the EGFR band in COS1 cells. DOI: http://dx.doi.org/10.7554/eLife.11835.018

Article Snippet: EGFR phosphosite-specific Westerns were performed using the following antibodies: pY845 (SCBT, #sc-575442), pY974 (CST, #2641S), pY992 (CST, #2235P), pY1045 (CST, #2237P), pY1068 (CST, #3777P), pY1086 (CST, #2220S), and pY1173 (CST, #4407S).

Techniques: Binding Assay

Journal: BMC cancer

Article Title: Possible predisposition for colorectal carcinogenesis due to altered gene expressions in normal appearing mucosa from patients with colorectal neoplasia.

doi: 10.1186/s12885-019-5833-8

Figure Lengend Snippet: Fig. 1 Simplified schematic illustration of pathways for Wnt/β-catenin, ERK/MAPK and PI3K/Akt and PGE2-metabolism. A) Canonical Wnt/β-catenin signaling. The engagement of the Wnt receptor, Frizzled, leads to the inhibition of the β-catenin destruction complex, composed of APC, axin and GSK3β. β-catenin thereby avoids ubiquitination and subsequent degradation, thus allowing it to translocate to the nucleus to activate an array of regulatory genes. B) The RAS/RAF/MEK/ERK MAPK pathway. Stimulation of the receptor tyrosine kinase (RTK) or G-protein coupled receptors (GPCRs) leads to sequential activation of RAS, RAF, MEK, and ERK causing modification of substrates promoting cell survival and proliferation. C) In the PI3K/Akt pathway, activation of the RTK or GPCRs leads to sequential modification of phosphatidyl inositol residues of the phospholipid bilayer. In this process, PI3K generates PIP3. PIP3 in association with PDK1 activates Akt. Akt then modulates the activity of downstream substrates including mTOR, thus promoting proliferation and cell survival. D) PGE2-metabolism. PGE2-synthesis begins with catalytic hydrolysis of membrane phospholipids by cytoplasmic phospholipase A2 (cPLA2), thus releasing arachidonic acid (AA). By the action of the COX-1 and COX-2, AA is converted to prostaglandin H2 (PGH2). PGH2 is then converted to PGE2 by prostaglandin E synthase (PTGES). The main exporter of PGE2 is thought to be multi-drug resistance related polypeptide 4 (MRP4). Removal of PGE2 from the extracellular compartment around target cells occurs by diffusion to the blood stream and subsequent uptake and degradation in lung, liver or kidney endothelial cells or by import to colonic epithelial cells through the prostaglandin transporter (PGT) and subsequent degradation by 15-prostaglandin dehydrogenase (15-PGDH). Through autocrine and paracrine signaling, extracellular PGE2 stimulates the prostaglandin receptors EP1–4. The EPs are GPCRs with EP1 being Gαq-coupled while EP2 and EP4 are Gαs-coupled. EP3 is capable of coupling with different G-proteins including Gαi, Gαs and Gαq

Article Snippet: In addition, attempts to detect cytoplasmic phospholipase A2 alpha (cPLA2A) using two different antibodies (Santa Cruz Biotechnology, sc-454 and Cell Signaling 2832) and protein phosphatase 2 isoform beta of scaffold subunit A (PPP2R1B) using mouse anti-PPP2R1B (Santa Cruz Biotechnology, sc-13, 600) proved unsuccessful in both Western blots and IHC.

Techniques: Inhibition, Ubiquitin Proteomics, Activation Assay, Modification, Activity Assay, Membrane, Diffusion-based Assay

Journal: The Journal of Experimental Medicine

Article Title: GSAP regulates lipid homeostasis and mitochondrial function associated with Alzheimer’s disease

doi: 10.1084/jem.20202446

Figure Lengend Snippet: GSAP and its binding proteins are involved in novel biological pathways. (A) Schematic of the experimental design to characterize the GSAP interactome. HA-EV was used as a negative control. (B) GO pathway enrichment analysis for GSAP-binding proteins. Top 20 significantly enriched pathways (P < 0.05) are shown based on P value (dot color) and gene count (dot size). (C) KEGG biological process enrichment analysis for GSAP-binding proteins. Top 20 significantly enriched pathways (P < 0.05) are shown based on P value (dot color) and gene count (dot size). (D) Volcano plot showing differentially enriched proteins (detailed in the methods) in HA-GSAP versus HA-EV co-IP MS experiments in N2a cells. GSAP itself (purple), proteins involved in trafficking (blue), and phosphorylation (red) are highlighted. FC, fold change. (E) Venn diagram showing overlapped protein between different lists. The circle area is not proportional to the sample size. (F) Meta-enrichment analysis of common GO biological pathways shared by two GSAP-binding protein lists. (G) Co-IP validation of GSAP interaction with PP1 and δ-COP (Arcn1) in HEK293T or N2a cells, respectively, via transient transfection. Representative data of three experiments.

Article Snippet: HEK293T cells (ATCC; CRL-11268) and HEK293-APP WT and GKO cells were grown in DMEM containing 10% FBS ( ).

Techniques: Binding Assay, Negative Control, Co-Immunoprecipitation Assay, Phospho-proteomics, Biomarker Discovery, Transfection

Journal: The Journal of Experimental Medicine

Article Title: GSAP regulates lipid homeostasis and mitochondrial function associated with Alzheimer’s disease

doi: 10.1084/jem.20202446

Figure Lengend Snippet: GSAP-binding protein and antibody validation. (A) Heatmap showing GSAP and binding protein levels in bait-expressing (HA-GSAP) versus EV (empty vector expression) samples in N2a co-IP and MS analyses. Proteins enriched in HA-GSAP samples are shown; mitochondrial proteins are highlighted in red. (B) GO biological process association for GSAP from experimental data and computational prediction (humanbase database; http://hb.flatironinstitute.org/gene/54103 ). *, based on previous experimental data. (C) GSAP-binding proteins identified through Y2H were visualized by the STRING App in Cytoscape. (D) Co-IP analysis of GSAP (HA-tagged) interaction with PHB (Flag-tagged) using Flag antibody. Representative data of two experiments. (E) HA-tagged human GSAP plasmid was transfected into HEK293T cells together with control (C) or GSAP siRNA. 48 h after transfection, cell lysates were collected and subjected to SDS-PAGE and immunoblot analysis. GSAP antibody from Thermo Fisher Scientific (Thermo) or R&D Systems (R&D) was used to detect GSAP. Representative data of two experiments.

Article Snippet: HEK293T cells (ATCC; CRL-11268) and HEK293-APP WT and GKO cells were grown in DMEM containing 10% FBS ( ).

Techniques: Binding Assay, Biomarker Discovery, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Transfection, Control, SDS Page, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: GSAP regulates lipid homeostasis and mitochondrial function associated with Alzheimer’s disease

doi: 10.1084/jem.20202446

Figure Lengend Snippet: GSAP interacts with Fe65 to regulate APP phosphorylation and trafficking. (A) Immunoblot analysis of protein levels in N2a695 cells transfected with control or GSAP siRNA (left panel). Quantification of APP phosphorylation at Thr668 normalized to total APP level (right panel). Data represent mean ± SEM; unpaired t test, **, P < 0.01. pT668, phospho-Thr668. Representative data of four experiments. (B) Co-IP analysis of full-length GSAP (HA-tagged) interaction with full-length Fe65 (Flag-tagged) using Flag antibody in HEK293T cells. Representative data of two experiments. (C) Co-IP analysis of GSAP C-terminal 16K domain (HA-tagged) coprecipitation with full-length Fe65 (Flag-tagged) using a Flag antibody in HEK293T cells. Representative data of two experiments. (D) Co-IP analysis of endogenous Fe65 interaction with GSAP and PP1 using Fe65 antibody in HEK293T cells. GSAP was detected using an antibody from R&D Systems. Representative data of two experiments. (E) Genomic DNA from CAD WT and Fe65KO cells was isolated, and PCR-amplified fragments flanking the CRISPR-Cas9 cleavage site were generated. PCR fragments were cloned into TOPO vector for Sanger sequencing. A 1-bp insertion (red) and deletion (blue) was identified in Fe65KO CAD cells (upper panel). Immunoblot analysis of proteins from WT and Fe65KO CAD cells (lower panel). (F) Immunoblot analysis of protein levels in CAD cells transiently overexpressing APP (left panel). Quantification of APP phosphorylation at Thr668 normalized to total APP level (right panel). Data represent mean ± SEM; unpaired t test, *, P < 0.05. Representative data from two experiments. (G) Representative confocal microscopy of Fe65 (red) and APP (green) localization in differentiated CAD cells. Arrow denotes the structure of Golgi apparatus. Scale bar, 5 µm. Representative data of ten cells. A.U., arbitrary units. (H) Maximum intensity projection of Airyscan Z-stack of WT (top left) and Fe65KO (top right) CAD cells from 95 slices and 0.173-µm step size and generated in Imaris. Scale bars, 5 µm. The images are representative of four independent experiments. WT (bottom left) and Fe65KO (bottom right) trajectories corresponding to the representative time-lapse image series are shown in the top panel and were reconstructed in MATLAB. Trajectory minimum cutoff time is 10 s. (I) Violin plots showing the velocity (left) and diffusion coefficient (right) distributions of single APP-GFP vesicles in WT and Fe65KO CAD cells. The median value is shown as the horizontal line in the box. The box presents interquartile range. The distributions were compared using the Mann–Whitney U test (**, P < 0.001; WT V median = 1.016 µm/s, KO V median = 1.038 µm/s; WT D median = 0.0187 μm2/s, and KO D median = 0.0290 μm2/s). (J) Co-IP analysis of GSAP (HA-tagged) with APP-C99 (Flag-tagged) in WT and Fe65KO (FKO) CAD cells. Representative data of two experiments. (K) Schematic of protein domain interactions within the APP–Fe65–GSAP complex. AICD, APP intracellular domain.

Article Snippet: HEK293T cells (ATCC; CRL-11268) and HEK293-APP WT and GKO cells were grown in DMEM containing 10% FBS ( ).

Techniques: Phospho-proteomics, Western Blot, Transfection, Control, Co-Immunoprecipitation Assay, Isolation, Amplification, CRISPR, Generated, Clone Assay, Plasmid Preparation, Sequencing, Confocal Microscopy, Diffusion-based Assay, MANN-WHITNEY