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: 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: 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: 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: 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: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: Probucol inhibits cholesterol efflux differently in NF cells vs F cells. The effect of a fixed probucol dose on apoA-I cholesterol efflux capacity in THP-1 non-foam (A) and foam cells (B). THP-1 cells were radiolabeled with [3H]cholesterol (48 h) or [3H]cholesterol, AcLDL (50 µg/ml) and 1% FBS. Cells were incubated for 48 h and treated with 10 µM 9cRA and 5 µg/ml 22-OH for 18 h as indicated in “Methods.” Cholesterol efflux was then initiated by the addition of apoA-I at the indicated doses for 4 h. The fractional cholesterol efflux is plotted as a function of acceptor concentration. In all experiments, efflux of [3H]cholesterol is expressed as mean±SD of triplicate measurements and represents three experiments. C. Concentration-dependent effect of probucol on apoA-I-mediated cholesterol efflux in THP-1 and J774 non foam cells respectively. Monolayers were radio labeled for 48 h as described in “Methods.” THP-1 and J774 cells were then equilibrated with 0.2% BSA and incubated with 10 µM 9cRA and 5 µg/ml 22-OH or cAMP (0.3 mM) for 18 h respectively. Monolayers were then incubated in the presence of increasing concentrations of probucol for 2h. After probucol treatment, cells were washed and incubated with RPMI containing 25 µg/ml lipid-free apoA-I for 4 h. Data are from a representative experiment with triplicate wells (n=3). Values are expressed as means + S.D. D. Efflux to apoA-I in the presence of probucol is almost abolished, in the BHK expressing ABCA1 cell type. BHK cells were grown as described in “Methods.” ABCA1-expressing BHK cells were radio labeled with [3H]cholesterol for 48 h in 1% FCS. To induce expression of ABCA1, BHK cells were incubated with 10 nM mifepristone containing 0.1% BSA for 18–20 h. Cell were treated with probucol for 2 h, washed and incubated with apoA-I for 24 h. Insert represents specific cholesterol efflux after subtracting efflux to BSA (0.2%) including diffusion. Results shown are representative of three independent experiments.

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Incubation, Concentration Assay, Labeling, Expressing, Diffusion-based Assay

Journal: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: Probucol results in less efflux inhibition in foam cells than non-foam cells. (A, B, C and D) Monolayers of J774 cells (A, B) and THP-1 cells (C, D) were labeled with 2 μCi/mL [3H]cholesterol (non-foam, A, C) or [3H]cholesterol and AcLDL (foam, B, D; 50 µg/ml), for 48 h in RPMI medium 1640 with 1% FCS. Cells were then incubated for 18 h with 0.2% BSA or not in the presence (solid bars) or absence (hatched bars) of 0.3 mM cpt-cAMP followed by incubation with or without 10 μM probucol as described in “Methods.” After 2 h of probucol treatment, cells were washed and then incubated with RPMI medium 1640 containing 25 μg/ml lipid-free apoA-I for 4 h. Data are from a representative experiment with triplicate wells (n=3). Values are expressed as mean ± SD. *P < 0.05 by Student's t-test.

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Inhibition, Labeling, Incubation

Journal: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: Macrophage foam cells are less sensitive to probucol-mediated inhibition of cholesterol efflux than non-foam cells.

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Inhibition

Journal: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: The effect of probucol on cholesterol efflux in foam cells without ABCA1 expression. Probucol produces a lesser degree of efflux inhibition in non-cAMP stimulated J774 foam cells vs non-foam cells. Monolayers were labeled with 2 μCi/ml [3H]cholesterol (A) or [3H]cholesterol and AcLDL (B, 50 µg/ml), for 48 h in RPMI medium 1640 with 1% FCS. Cells were then incubated for 18 h or not with 0.2% BSA in the absence of 0.3 mM cpt-cAMP followed by incubation with 10 μM probucol for 2 h. Cells were washed and then incubated with medium containing 25 μg/ml lipid-free apoA-I for 24 h. C. Probucol completely inhibits cholesterol efflux in the absence of ABCA1 expression in non-mifepristone induced BHK cells. BHK cells were labelled with [3H]cholesterol 48 h, 1% FCS. Afterwards, cells were incubated in DMEM containing 0.2% BSA for 18–20 h. This was followed by an incubation with 10 μM probucol for 2 h. Non-induced BHK cells were washed and incubated with medium containing 10 μg/ml lipid-free apoA-I for 24 h. BHK cells incubated alone were used as control. Inset represents specific cholesterol efflux inhibited by probucol obtained after subtracted cholesterol efflux from background.D. Non-stimulated J774 and BHK cells do not express ABCA1 transporter protein. Cells were grown as described in Materials and methods, cells were lysed at 4°C with 20 mM Tris, 5 mM EDTA, and 5 mM EGTA; pH 7.5 containing 0.5% n-dodecylmaltoside. Protein concentration was determined by standard assay (Bio-Rad). Cells were separated by SDS-PAGE (4–22.5%) and immunoblotted using antibodies against human ABCA1 and the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The molecular weight (Bio-Rad) is shown on the right of the gel. Data are from a representative experiment with triplicate wells (n=3). Values are expressed as mean±SD. *P < 0.05 by Student's t-test. NF; non foam, F; foam

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Expressing, Inhibition, Labeling, Incubation, Control, Protein Concentration, SDS Page, Molecular Weight

Journal: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: Probucol inhibits cholesterol efflux in J774 macrophages non-expressing ABCA1.

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Inhibition

Journal: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: Probucol does not alter ABCA1 nor ABCG1 protein expression in foam cells and non-foam cells. Cells were treated as described above, and after probucol incubation they were washed with PBS and solubilized and separated by (4-22.5%) SDS-PAGE. (A, D) ABCA1 from J774 and THP-1 cells lysis was detected by anti-ABCA1 antibody respectively. (B, C, and D) Changes in ABCA1 protein expression were determined by normalizing against the densitometric intensity of GAPDH. (E) Human ABCG1-overexpressing CHO-K1 cells were labeled with 1 µCi/ml [3H]cholesterol for 24 h, washed, and then equilibrated for 2 h in the presence of absence of different concentration of probucol. Efflux was promoted for 6 h to 12.5 µg/ml of HDL. (F) Probucol treatment did not significantly affect ABCG1 protein expression in THP-1 foam cells. ABCG1 from THP-1 cells lysis was detected by anti-ABCG1 antibody. Changes in ABCA1 protein expression were determined by normalizing against the densitometric intensity of GAPDH. Each sample was run in triplicate. Values are expressed as means ± SD. GAPDH was used as a loading control. *P < 0.05 by Student's t-test.

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Expressing, Incubation, SDS Page, Lysis, Labeling, Concentration Assay, Control

Journal: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: Characterization of lipidated apoA-I-containing particles generated from pretreated THP-1 cells with probucol. Monolayers were labeled with 2 μCi/ml [3H]cholesterol for 48 h in RPMI medium 1640 with 1% FCS. After 2 h of probucol treatment, cells were washed and then incubated with RPMI medium 1640 containing 10 μg/ml lipid-free apoA-I for 24 h. Afterwards, the media were collected and prepared as described in “Experimental Procedures.” Samples were separated by 2D-PAGGE and apoA-I was detected by anti-apoA-I antibody as described in “Methods.” (A, B): J774 non-foam and foam cells whole medium. (C, D): Human THP-1 macrophage non foam and foam cells whole medium. Molecular size markers (diameter in nm) are indicated. The HDL particle diameters are derived from the relative mobility values of the centers of the various bands. Molecular weight markers were revealed by Ponceau S. (E, F). Nascent HDL (nHDL) particles formed from foam cells pretreated with probucol contain more lipids than those from non foam cells. radioactivity applied to gel electrophoresis. Radioactivity appearing in gels corresponding to apoA-I and apoA-I + probucol was counted as described in “Methods.” [3H]cholesterol (E) from foam cells versus (F) non foam cells in the presence of apoA-I or apoA-I + probucol was expressed as a percentage of control (100%, in the absence of apoA-I). Results shown are representative of three independent experiments. *P < 0.05 by Student's t-test.

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Generated, Labeling, Incubation, Derivative Assay, Molecular Weight, Radioactivity, Nucleic Acid Electrophoresis, Control

Journal: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: Dynamics of nascent HDL generated from foam cells in the presence of probucol. Radiolabelled [3H]cholesterol loaded nascent HDL (nHDL)-like lipoprotein apoA-I derived from THP-1 foam cells pretreated with or without probucol were incubated with normolipidemic human plasma for 6 h at 37°C. After incubation, apoB was precipitated with 50/50 vol/vol 20% PEG 6000. Apo B containing particle fractions were dialyzed and [3H]cholesterol transfer was calculated between nHDL and apo B fractions as indicated (A). Counts were made in triplicates. B. Probucol effect on free cholesterol oxidation by cholesterol oxidase in J774 mouse macrophages (C) and in THP-1 non-foam and foam macrophages. Monolayers were labeled with 3 μCi/ml [3H]cholesterol for 48 h with 1% FCS or with 3 μCi/ml [3H]cholesterol, AcLDL (50 µg/ml) and 1% FCS for 48 h with 1% FCS. Cells were treated with probucol as described above, but the incubation with cAMP was done with 10 μM 9cRA and 5 μg/ml 22-OH. Data are from a representative experiment with triplicate wells (n=3). Values are expressed as mean±SD. *P < 0.05 by Student's t-test. Control cells (CTR) shows the results from non-treated cells, while CTR-probucol (PB) shows the results from cells treated with probucol 10 mM for 2h.

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Generated, Derivative Assay, Incubation, Clinical Proteomics, Labeling, Control

Journal: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: Macrophage foam cells are more sensitive to probucol-mediated decrease of the total accessible plasma membrane pool of cholesterol than non-foam cells.

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Clinical Proteomics, Membrane, Inhibition

Journal: BBA Advances

Article Title: Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

doi: 10.1016/j.bbadva.2021.100003

Figure Lengend Snippet: Summary of main findings.

Article Snippet: Mouse-derived peritoneal macrophages J774 cells were purchased from (ATCC TIB-67, Cedarlane, Burlington, Ontario, Canada).

Techniques: Expressing, Functional Assay, Generated, Transferring

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: Identification of HAPLN1 as a factor capable of inducing NF-κB activity in myeloma cells. A, SDS-PAGE and Coomassie Blue staining of the enriched fractions with NF-κB–inducing activity previously identified by Markovina et al. (35). Three bands (*) from fraction 3 (F3) that contained the highest NF-κB–inducing activity were excised and analyzed by nano-LC-MS/MS. Shown are identified factors with the number of unique peptides identified in parentheses. B, EMSA analysis of RPMI8226 cells incubated with 10 ng/ml TNFα for 15 min or CM collected from HEK293 transiently transfected with vector containing C-terminally MYC-tagged proteins: calumenin, osteonectin, or galectin-3–binding protein (Gal-3BP) for 2 h. Results are representative of at least three independent experiments. C, representative immunoblot analysis (IB), using anti-MYC antibody, of CM from HEK293 cells transiently transfected with calumenin, osteonectin, or galectin-3–binding protein expression vector showing the presence of corresponding secreted factors. D, immunoblot analysis, using anti-MYC antibody, of HAPLN1 expression in CM collected from transiently transfected HEK293 cells with the HAPLN1 expression vector positive for induction of NF-κB activity. Smaller HAPLN1 (H1) fragments are indicated *, larger immunoreactive bands. E, EMSA analysis of RPMI8226 cells treated with CM from transiently transfected HEK293 cells containing HAPLN1 for 2 h in the absence or presence of 100 nm bortezomib (Bort). Induced NF-κB activity is highly variable in more than five independent experiments, possibly dependent on the variability of smaller H1 species seen in D.

Article Snippet: Cell line culture RPMI8226, MM.1S, NCI-H929, and HEK293 cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Activity Assay, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Incubation, Transfection, Plasmid Preparation, Binding Assay, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: Generation and characterization of recombinant HAPLN1 proteins. A, diagram of HAPLN1 domains. The numbers indicate the amino acid positions. SP, signal peptide; IG, immunoglobulin-like domain; PTR1, proteoglycan tandem repeat 1 domain; PTR2, proteoglycan tandem repeat 2 domain. B, SDS-PAGE and GelCodeTM staining of GST-fused HAPLN1 domain proteins. Full-length (FL) HAPLN1 (not GST-tagged) is from R&D. *, band of interest. C (top), representative EMSA analysis of NF-κB and Oct-1 activities in RPMI8226 cells following incubation with FL-H1, GST-fused H1-IG, H1-P1, and H1-P2 at 100 nm for 4 h or TNFα (10 ng/ml) for 15 min. Bottom, graph represents results of the mean -fold NF-κB activation ± S.D. (error bars) of three independent EMSA experiments shown above. *, p < 0.05; **, p < 0.01 when compared with appropriate control (untreated or GST only). D, representative EMSA analysis of three independent experiments where RPMI8226 cells were stimulated with increasing doses of lipopolysaccharide (LPS) for 1 h or 10 ng/ml TNFα for 15 min. E, representative graph of three independent endotoxin quantifications using the Pierce LAL chromogenic endotoxin quantification kit (Thermo Fisher Scientific). Depicted is the span of the average amount of endotoxin found in three independent H1-P1 purifications. The eluted fractions were further diluted 50–100-fold when added to culture media for NF-κB activation analyses, making the levels of contaminating lipopolysaccharide at least 3 orders of magnitude below what is detectable for NF-κB activation by EMSA.

Article Snippet: Cell line culture RPMI8226, MM.1S, NCI-H929, and HEK293 cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Recombinant, SDS Page, Staining, Incubation, Activation Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: HAPLN1-PTR1 domain activates NF-κB in myeloma cells. A (top), representative EMSA analysis of RPMI8226 cells incubated with increasing concentrations of H1-P1 for 2 h. Bottom, graph represents results of the mean -fold NF-κB activation ± S.D. (error bars) of three independent EMSA experiments (above). *, p < 0.05; **, p < 0.01 when compared with untreated (0 nm). B (top), representative EMSA analysis of RPMI8226 cells incubated with 100 nm H1-P1 for the times indicated. Bottom, graph represents results of the mean -fold NF-κB activation ± S.D. of three independent EMSA experiments (above). *, p < 0.05; **, p < 0.01 when compared with unstimulated 1-h lane. C, supershift analysis of RPMI8226 cells treated with 100 nm H1-P1 for 2 h using antibodies against the five NF-κB family members. Results are representative of three independent experiments. D, SDS-PAGE and GelCodeTM staining of GST-fused H1-P1 following incubation with GST-fused PreScission protease. H1-P1c, cleaved H1-P1 fragment. E, EMSA analysis of RPMI8226 cells pretreated for 30 min with 100 nm bortezomib as indicated and stimulated with 100 nm H1-P1c or 10 ng/ml TNFα for the times indicated, representative of two independent experiments. F, RPMI8226 cells treated with the indicated concentrations of H1(32–354) for 4 h and analyzed by EMSA, representative of two independent experiments. G, RPMI8226, MM.1S, and H929 cell lines were incubated with 10 ng/ml TNFα for 15 min or 100 nm H1-P1 for 4 h and assayed for induction of NF-κB activity by EMSA, representative of three independent experiments. Where indicated, EMSA was done in the presence of a RelB antibody (RelB Aby) to shift RelB complexes to enable quantification of canonical NF-κB activation. -Fold change of NF-κB DNA binding as measured by phosphor image quantification, corrected for Oct-1 DNA binding control, and normalized to unstimulated but RelB antibody–treated, is indicated below the gel.

Article Snippet: Cell line culture RPMI8226, MM.1S, NCI-H929, and HEK293 cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Incubation, Activation Assay, SDS Page, Staining, Activity Assay, Binding Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: Summary of H1-P1 induction of NF-κB activity in different cell lines of different cell types Cells were treated with 100 n m H1-P1 for 4 h as analyzed by EMSA. +, a positive induction of NF-κB activity; −, no induction of NF-κB activity; +/−, a very weak induction of NF-κB activity. MM, multiple myeloma; MCL, mantle cell lymphoma; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia.

Article Snippet: Cell line culture RPMI8226, MM.1S, NCI-H929, and HEK293 cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: Hyaluronic acid binding is unnecessary for HAPLN1-PTR1–induced NF-κB activation. A, EMSA analysis of NF-κB activity in RPMI8226 cells following incubation with low-molecular weight HA (LMW-HA; 29 kDa) or high molecular weight HA (HMW-HA; 289 kDa) at the microgram amounts indicated for 2 h. Results are representative of two independent experiments. B, EMSA analysis of RPMI8226 cells treated with 100 nm H1-P1, low-molecular weight HA, high-molecular weight HA, or hyaluronidase (HYAL) in the amounts indicated for 4 h. C, H1-P1 HA-binding mutant (H1-P1 HABD mt) was made by mutating the critical residues necessary for HA binding within H1-P1 to alanine where illustrated. RPMI8226 cells were incubated with the indicated concentrations (nm) of recombinant GST-fused H1-P1 HABD mt for 4 h and assayed by EMSA. Results are representative of three independent experiments. -Fold change of NF-κB activity was determined as described previously.

Article Snippet: Cell line culture RPMI8226, MM.1S, NCI-H929, and HEK293 cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Binding Assay, Activation Assay, Activity Assay, Incubation, Molecular Weight, High Molecular Weight, Mutagenesis, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: HAPLN1-PTR1 causes bortezomib-resistant NF-κB activation. A, representative EMSA analysis of RPMI8226 cells incubated with 10 ng/ml TNFα or 100 nm H1-P1 in the absence or presence of increasing concentrations (nm) of bortezomib (Bort). B, graph depicts the mean ± S.D. (error bars) of the quantification of three independent replicates of EMSA analysis as in A. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, the proteasome activity in RPMI8226 cells was analyzed following treatment with increasing doses of bortezomib (nm) in the presence and absence of 100 nm H1-P1 using a Proteasome-Glo cell-based assay (Promega) and plotted using the basal proteasome activity in 0 nm bortezomib set as 1. Results represent mean ± S.D. of three independent experiments. D, EMSA analysis of RPMI8226 cells incubated with increasing concentrations of lactacystin (μm) and stimulated with 10 ng/ml TNFα for 15 min or 100 nm H1-P1 for 4 h. Results are representative of two independent experiments. E, EMSA analysis of RPMI8226 cells pretreated with 100 nm bortezomib for 30 min and stimulated with increasing doses of TNFα (ng/ml) for 15 min. -Fold change of NF-κB activity was determined as described previously.

Article Snippet: Cell line culture RPMI8226, MM.1S, NCI-H929, and HEK293 cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Activation Assay, Incubation, Activity Assay, Cell Based Assay

Journal: The Journal of Biological Chemistry

Article Title: Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma

doi: 10.1074/jbc.RA117.000667

Figure Lengend Snippet: HAPLN1-PTR1 causes bortezomib-resistant IκBα degradation. A, representative immunoblot analysis of IκBα, IκBβ, and tubulin in RPMI8226 cells pretreated for 30 min with cycloheximide (Cx; 20 μg/ml) or bortezomib (Bort) (100 nm) and stimulated with H1-P1 (100 nm) where indicated. B, three biological replicates of degradation of IκBα and IκBβ as in A were quantified and plotted with mean ± S.D. (error bars). *, p < 0.05. C, immunoblot analysis of RPMI8226 cells pretreated for 30 min with 100 nm bortezomib where indicated and stimulated with 10 ng/ml TNFα for 15 min or 100 nm H1-P1 for 2 h. The top gel image is a longer exposure of the IκBα blot. The positions of IκBα, phospho-IκBα, and IκBα ubiquitin ladders are indicated. Results are representative of at least three independent experiments. D, RPMI8226 cells pretreated for 30 min with 10 μm IKK16 and 20 μg/ml cycloheximide were stimulated with H1-P1 for the indicated times and analyzed by EMSA and immunoblotted (IB) for indicated proteins. Results are representative of three independent experiments. E, EMSA and immunoblot analysis for the indicated proteins of RPMI8226 cells pretreated for 30 min with increasing concentrations MLN4924 (μm) and stimulated with 100 nm H1-P1 for 4 h or 10 ng/ml TNFα for 15 min and analyzed by EMSA and immunoblotting for the indicated proteins. F, results represent mean ± S.D. (*, p < 0.05; **, p < 0.01) of quantification of three independent EMSA and phospho-IκBα immunoblot analyses as in E at 100 μm MLN29424.

Article Snippet: Cell line culture RPMI8226, MM.1S, NCI-H929, and HEK293 cell lines were obtained from American Type Culture Collection (ATCC).

Techniques: Western Blot, Ubiquitin Proteomics

Journal: iScience

Article Title: M-Motif, a potential non-conventional NLS in YAP/TAZ and other cellular and viral proteins that inhibits classic protein import

doi: 10.1016/j.isci.2025.112105

Figure Lengend Snippet: Characterization of structural requirements and functional properties of TAZ and YAP nuclear import (A) Domain organization of TAZ with regions important for nucleocytoplasmic shuttling highlighted. Lats-phosphorylation sites are shown as yellow circles with labels, hydrophobic motifs FLxx[I,V,L,M] as blue lines. CC: coiled-coil region; NES: nuclear export signal; NLS: nuclear localization signal; PBM: PDZ domain binding motif; TAD: transactivation domain; TBD: TEAD-binding domain; WW: WW domain; WW-NLS: globular NLS composed of the WW domain. (B) Localization of diffusion-limited 5C constructs in pig proximal tubular epithelial LLC-PK1 cells, also see Figure S1 . 5C-R5A-NES: shuttling control comprising a variant of the SV40 NLS and the HIV Ref NES. WT: 5C-TAZ 4SA or 5C-YAP 5SA; ΔNLS: constructs with deletion of residues 321–345 (TAZ) or 413–427 (YAP); ΔPBM: constructs missing the last 5 aa (the PBM); control: 5C control. Left: Representative fluorescence microscopy images of transfected cells, without (top) and with 4h LMB treatment (bottom). The scale bar represents 50 μm. Right: Quantification of the nuclear accumulation of the 5C-constructs upon LMB treatment for the indicated times. Median nucleocytoplasmic fluorescence ratios (median N/C of >100 cells) were determined for individual constructs as described previously and data were fitted to mono-exponential growth curves with plateau. Number of repeats: 4. (C) Localization of 5C-NLS constructs. Left: Representative fluorescence microscopy images of transfected LLC-PK1 cells. The scale bar represents 50 μm. Right: Quantification of the nuclear accumulation of 5C, 5C-TAZ NLS, 5C-YAP-NLS, and 5C-R5A in LLC-PK1 and HEK cells. (D) The TAZ NLS inhibits the nuclear uptake of full-length TAZ. Cells were transfected with constant amounts of 5C or 5C-TAZ 4SA in combination with mCherry-TAZ NLS at ratios 1:0, 1:1, 1:2, 1:3, and 1:4. mCherry encoding vector was added to keep the total amounts of DNA constant. The increase in nuclear accumulation of 5C constructs after 6h LMB treatment, relative to no LMB addition is shown as ΔN/C (LMB). Number of repeats: 8. Also see Figure S2 A. (E) Localization of 1C constructs in LLC-PK1 and RPE-1 cells. (F and G) Interaction between TAZ and IPO7. mCitrine (1C) and 1C-TAZ constructs were expressed in HEK cells. IPO7 (F) or Citrine-constructs (G) were immunoprecipitated and analyzed by western blotting using GFP- and IPO7 specific antibodies. Red asterisks indicate the position of relevant bands. (H) Effect of ivermectin (iver; 25 μM) on the nuclear accumulation of endogenous TAZ/YAP and MRTF upon treatment with low calcium medium (LCM). Response is given as the percentage of uninhibited N/C increase at 60 min (TAZ/YAP) or 20 min (MRTF) LCM treatment. (I) Nuclear accumulation of 5C-TAZ NLS and 5C-SV40 NLS in the presence of indicated concentrations of ivermectin. Also see Figure S2 B. (J) Effect of ivermectin on the nuclear accumulation of shuttling 5C-constructs induced by LMB treatment. ΔN/C (LMB) are calculated as in (D). (K) Nuclear import of TAZ 4SA is Ran-independent. Cells were co-transfected with indicated constructs and non-related (siNR) or Ran-specific (siRan) siRNA and treated with LMB for 4h. (L) Nuclear import of TAZ 4SA is ATP-independent. Left: Representative fluorescence microscopy images showing the effect of 2-deoxyglucose and azide on the cellular distribution of KPNA2. The scale bar represents 50 μm. Right: cells transfected with indicated constructs and treated for 4h with or without LMB, in the presence or absence of 2-deoxyglucose and azide. Means ± SD are depicted, and number of repeats are indicated. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ∗∗∗∗ p < 0.001; one-way ANOVA and Tukey-Kramer test.

Article Snippet: These cells were cultured in high-glucose DMEM. hTERT RPE cells, a Telomerase immortalized human retina pigmented epithelial cell line (female) were obtained from the American Type Culture Collection (ATCC Cat# CRL-4000, RRID:CVCL_4388).

Techniques: Functional Assay, Phospho-proteomics, Binding Assay, Diffusion-based Assay, Construct, Control, Variant Assay, Fluorescence, Microscopy, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: iScience

Article Title: M-Motif, a potential non-conventional NLS in YAP/TAZ and other cellular and viral proteins that inhibits classic protein import

doi: 10.1016/j.isci.2025.112105

Figure Lengend Snippet:

Article Snippet: These cells were cultured in high-glucose DMEM. hTERT RPE cells, a Telomerase immortalized human retina pigmented epithelial cell line (female) were obtained from the American Type Culture Collection (ATCC Cat# CRL-4000, RRID:CVCL_4388).

Techniques: Recombinant, Transfection, Luciferase, Reporter Assay, Control, Plasmid Preparation, Software

Journal: Biology Open

Article Title: Myosin 1E localizes to actin polymerization sites in lamellipodia, affecting actin dynamics and adhesion formation

doi: 10.1242/bio.20135827

Figure Lengend Snippet: (A) Domain organization of Myosin 1 isoform. (B–F) Photoactivation by 405 nm laser in the ROA (white circle) and observation by 561 nm laser after 2 sec, of (B) Plasma membrane-PAGFP membrane diffusion control, (C) PAmcherry-Myosin 1B, (D) 1G, (E) 1C, (F) 1E. Bar 2 µm, B–F is in same scale. (G) Normalized (against the intensity at ROA) average intensity perpendicular to the long axis of the 2 sec figure panels in Myosin 1C and Myosin 1E. (H) Profile and (I) kymograph of mApple-Myosin 1E and plasma membrane GFP (PM-GFP) in spreading cell. Arrows indicate Myosin 1E accumulation. (J) Quantitation of absolute intensity in Myosin 1E channel in (H) ( supplementary material Movie 1 ). Bar 5 µm. (K) Enlarged view of the boxed region in (H), showing spots in Myosin channel behind the lamellipodial tip.

Article Snippet: DNA construct containing Myosin1F (ATCC clone MGC-40199, I.M.A.G. clone id-5213035) and Myosin 1B (ATCC No-10699052, I.M.A.G. clone id-6821232 and ATCC No-10469400, I.M.A.G. clone id-6487332) were purchased from BioRev/BioGen®.

Techniques: Clinical Proteomics, Membrane, Diffusion-based Assay, Control, Quantitation 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