anti sdc4 apc  (R&D Systems)

Journal: Nature Communications

Article Title: Post-transcriptional control of a stemness signature by RNA-binding protein MEX3A regulates murine adult neurogenesis

doi: 10.1038/s41467-023-36054-6

Figure Lengend Snippet: a MEX3A RNA-immunoprecipitation (RIP) diagram. IP: immunoprecipitation; C: crosslinked samples; NC: non-crosslinked samples. b Protein-protein interaction network generated from MEX3A-bound coding RNAs (STRING PPI enrichment p -value = 3.67 × 10 −11 ). Line thickness indicates strength and confidence of the connection ( https://string-db.org/ ). See Supplementary Fig. and Supplementary Data for detailed target lists. c Representative Gene Ontology (GO, Biological Process) terms enriched in the subset of MEX3A-bound coding RNAs (see Supplementary Data for a complete GO list). Significance values are reported after FDR correction. d Scatterplot comparing differential expression (DE) data from DFFDA high vs. DFFDA low populations with MEX3A RIP-seq enrichment (FDR < 0.05). Red dots represent up-regulated, and blue dots down-regulated genes in DFFDA high cells. Dark red and blue dots label DE genes also bound by MEX3A. e Overlap between MEX3A-bound genes (orange) and genes enriched in DFFDA high (red, top) and DFFDA low (blue, bottom) populations. DFFDA high and MEX3A-bound genes show more overlap than expected ( p -value = 8.5 × 10 −8 , by hypergeometric test). f DE data from isolated pNSC vs. aNSC populations with MEX3A RIP-seq enrichment data (FDR < 0.05). Red dots represent up-regulated, and blue dots down-regulated genes in pNSCs. Dark red and blue dots label DE genes also bound by MEX3A. g Overlap between MEX3A-bound genes (orange) and genes enriched in pNSCs (red, top) and aNSCs (blue, bottom). pNSC and MEX3A-bound genes show more overlap than expected ( p -value=0.003, by hypergeometric test). Quantification of Aqp4 ( h ) and Sdc4 ( i ) gene expression by RT-qPCR in Mex3a +/+ and Mex3a KI/KI NSCs (left panels) ( * p -value < 0.05 and NS = 0.317 by unpaired two-tailed Student’s t -test). AQP4 ( h ) and SDC4 ( i ) protein levels (median fluorescence intensity) by FACS in wild-type and KI homozygous NSC cultures (right panels) (p-values: AQP4 = 0.032, SDC4 = 0.011, by unpaired two-tailed Student’s t -test). Box plots show median± interquartile range and whiskers define minimum to maximum. Exact p -values and the number of biologically independent samples used are indicated. Source data are provided as a Source Data file.

Article Snippet: Individual cells were incubated with 100 μl of the specific fluorescent-labeled primary antibodies (human anti-SDC4-APC (Miltenyi, cat. no. 130-109-831, 1:200); rabbit anti-AQP4 (Sigma, cat. no. HPA014784, 1: 150)) in flow cytometry blocking buffer at 4 °C for 30 min. Immunostained samples were analyzed in an LSR-Fortessa cytometer (350, 405, 488, 561 and 640 nm lasers, Becton Dickinson) with FACSDiva (v8.0.2, BD).

Techniques: Immunoprecipitation, Generated, Expressing, Isolation, Quantitative RT-PCR, Two Tailed Test, Fluorescence

Journal: International journal of molecular sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Figure 4. Effect of SDC4 knockdown (KD) or heparin inhibition on virus internalization into Calu-3 cells. SDC4 KD in Calu-3 cells was performed previously using a lentiviral vector specific to human SDC4. (A) SDC4 expression levels were measured with imaging flow cytometry, as shown by the representative histograms and cellular images. Detected SDC4 levels of KD cells were normalized to WT Calu-3 cells as standards. The bars represent the mean + SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05. (B,C) SDC4 KD and WT Calu-3 cells were exposed to 1 MOI of the heat-inactivated WT SCV2, Delta, and Omicron variants. For GAG inhibition, the viruses were preincubated with heparin (200 ug/mL for 30 min at 37 ◦C) before being added to the cells. (D,E) Representative flow cytometry histograms and cellular images show the intracellular fluorescence of WT or SDC4 KD Calu-3 cells treated with the viruses in the presence or absence of heparin. Scale bar = 20 µm. (F,G) Detected intracellular fluorescent signals were normalized to WT Calu-3 (F) cells or cells untreated with heparin (G) as standards. The bars represent the mean + SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis FlowSight, Luminex Corporation, Austin, TX, USA) with APCconjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A).

Techniques: Knockdown, Inhibition, Virus, Plasmid Preparation, Expressing, Imaging, Cytometry

Journal: International journal of molecular sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Figure 5. SDC4 binding of WT SCV2 and the Delta an Omicron variants. (A) SDS-PAGE showing SDC4 immunoprecipitated with an antibody specific for the spike’s amino acid sequence 1000-1200 from extracts of virus-treated Calu-3 cells. Lane 1: a total of 1 ug of recombinant SDC4; lanes 2–4: immunoprecipitates of Calu-3 cells treated with either WT SCV2, Delta, or Omicron, respectively; Lane 5: immunoprecipitate of untreated control Calu-3 cells. Standard protein size markers are indicated on the right. SDC4 signals were detected with UVITEC Alliance Q9 Advanced Imager, and the intensity of bands was analyzed with the NineAlliance© software. (B) Detected band intensities were normalized to WT SCV2-treated Calu-3 cells as standards. The bars represent the mean + SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ns: not significant.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis FlowSight, Luminex Corporation, Austin, TX, USA) with APCconjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A).

Techniques: Binding Assay, SDS Page, Immunoprecipitation, Sequencing, Virus, Recombinant, Control, Software

Journal: International journal of molecular sciences

Article Title: Exploring the Syndecan-Mediated Cellular Internalization of the SARS-CoV-2 Omicron Variant.

doi: 10.3390/ijms241814140

Figure Lengend Snippet: Figure 6. Effect of SDC4 KD on the cellular entry and gene delivery of WT SCV2, Delta, or Omicron PSVs in Calu-3 cells. SDC4 KD and WT Calu-3 cells were treated with either WT SCV2, Delta, or Omicron PSVs. (A,B) Representative cellular images and flow cytometry histograms showing the intracellular fluorescence of WT or SDC4 KD Calu-3 cells treated with the PSVs. Scale bar = 20 µm. (C) Detected fluorescence intensities were normalized to WT Calu-3 cells treated with the respective PSVs. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001.

Article Snippet: Stable KD cells were selected in 2 mg G418 and sorted using imaging flow cytometry (Amnis FlowSight, Luminex Corporation, Austin, TX, USA) with APCconjugated anti-SDC4 antibody (RnD Systems, Minneapolis, MN, USA, cat. no. FAB29181A) and respective isotype control (rat IgG2A APC isotype control, RnD Systems, cat. no. IC006A).

Techniques: Cytometry

Journal: Cell Reports Medicine

Article Title: Tissue-specific transcriptional profiles and heterogeneity of natural killer cells and group 1 innate lymphoid cells

doi: 10.1016/j.xcrm.2022.100812

Figure Lengend Snippet: Inter-tissue-specific signatures of NK and ILC1 subpopulations (A) Transcriptome-based clusters computed for each organ separately, projected onto the multi-organ integrated UMAP obtained with Harmony. (B) Heatmap representing overlap coefficient dissimilarity between transcriptome-based clusters calculated separately for each organ. (C) Dot plot of NK cell-specific versus ILC1-specific markers. Intermediate clusters were not taken into account. (D) Dot plot of specific markers for each cluster. (E) Flow cytometry profiles of CD49a, DNAM-1, PD-1H, and SDC4 expression, analyzed in Lin − NKp46 + NK1.1 + cells. Histograms represent the frequency of CD49a − CD49b + NK cells and CD49a + CD49b − ILC1s for each indicated marker across organs. Data are shown as mean ± SEM and are pooled from two independent experiments. Each point represents a pool of two mice.

Article Snippet: APC anti-mouse SDC4 (REA640) , Miltenyi Biotec , Cat# 130-109-831; RRID: AB_2653641.

Techniques: Flow Cytometry, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: Tissue-specific transcriptional profiles and heterogeneity of natural killer cells and group 1 innate lymphoid cells

doi: 10.1016/j.xcrm.2022.100812

Figure Lengend Snippet:

Article Snippet: APC anti-mouse SDC4 (REA640) , Miltenyi Biotec , Cat# 130-109-831; RRID: AB_2653641.

Techniques: Recombinant, Saline, Staining, Cell Isolation, Sensitive Assay, Software

Journal: International journal of molecular sciences

Article Title: Biodistribution and Cellular Internalization of Inactivated SARS-CoV-2 in Wild-Type Mice.

doi: 10.3390/ijms23147609

Figure Lengend Snippet: Figure 5. Effect of SDC4 knockdown (KD) on the internalization of inactivated SARS-CoV-2 into L929 and RAW cells. SDC4 KD in L929 and RAW cells was performed using a lentiviral vector specific to mouse SDC4. (A) Western blot validation of SDC4 kD in L929 and RAW cells. β-tubulin was used as a loading control. (B) SDC4 expression levels were also measured with imaging flow cytometry, as

Article Snippet: The SDC expression of the applied cell lines (K562 cells, SDC4 transfectants, L929, and RAW 264.7) was measured with flow cytometry by using APC-labeled anti-human SDC4 (R&D Systems, Minneapolis, MN, USA; SDC4: monoclonal rat IgG2a clone #336304, cat. no. FAB29181A) or primary anti-murine SDC antibodies (mouse SDC1, cat. no. AF3190, R&D Systems, Minneapolis, MN, USA; mouse SDC2, cat. no. PA5-95938, Invitrogen, Waltham, MA, USA; mouse SDC3, cat. no. PA5-47377, Invitrogen, Waltham, MA, USA; mouse SDC4-APC labeled antibody, cat. no. 130-109-831, Miltenyi Biotec, Bergisch Gladbach, Germany) and appropriate secondary antibodies (rabbit anti-goat IgG (H+L) cross-adsorbed secondary antibody, Alexa FluorTM 633, cat. no. A-21086, Invitrogen, Waltham, MA, USA; goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa FluorTM 633, cat. no. A-21070, Invitrogen, Waltham, MA, USA) and respective isotype controls according to the manufacturer’s protocol.

Techniques: Knockdown, Plasmid Preparation, Western Blot, Biomarker Discovery, Control, Expressing, Imaging, Flow Cytometry

Journal: International journal of molecular sciences

Article Title: Biodistribution and Cellular Internalization of Inactivated SARS-CoV-2 in Wild-Type Mice.

doi: 10.3390/ijms23147609

Figure Lengend Snippet: Figure 6. SARS-CoV-2 colocalizes with SDC4 during cellular entry. L929 and RAW cells were in- cubated with heat-inactivated SARS-CoV-2 (at 1 MOI) for 3 h at 37 ◦C. After incubation, the cells were washed, fixed, permeabilized, and treated with antibodies specific for the spike glycoprotein (AF 488 labeled) and SDC4 (APC labeled). Colocalization of SARS-CoV-2 with SDCs was analyzed with confocal microscopy. (A) Microscopic analyses of SARS-CoV-2 and SDC colocalization. Repre- sentative images of three independent experiments are shown. Scale bar = 10 µm. (B) The MOC and PCC ± SEM for the overlap and colocalization of SDC with SARS-CoV-2 (indicated below the images) were calculated by analyzing 18 images with an average of 12 cells in each image (from 3 separate samples). Scale bar = 10 µm. (C) A representative Western blot showing SDC4 immunoprecipitated with SARS-CoV-2 in L929 and RAW cells. Lane 1: 0.5 µg of SDC4; lanes 2–3: immunoprecipitates of SARS-CoV-2-treated L929 and RAW cells. Standard protein size markers are indicated on the right.

Article Snippet: The SDC expression of the applied cell lines (K562 cells, SDC4 transfectants, L929, and RAW 264.7) was measured with flow cytometry by using APC-labeled anti-human SDC4 (R&D Systems, Minneapolis, MN, USA; SDC4: monoclonal rat IgG2a clone #336304, cat. no. FAB29181A) or primary anti-murine SDC antibodies (mouse SDC1, cat. no. AF3190, R&D Systems, Minneapolis, MN, USA; mouse SDC2, cat. no. PA5-95938, Invitrogen, Waltham, MA, USA; mouse SDC3, cat. no. PA5-47377, Invitrogen, Waltham, MA, USA; mouse SDC4-APC labeled antibody, cat. no. 130-109-831, Miltenyi Biotec, Bergisch Gladbach, Germany) and appropriate secondary antibodies (rabbit anti-goat IgG (H+L) cross-adsorbed secondary antibody, Alexa FluorTM 633, cat. no. A-21086, Invitrogen, Waltham, MA, USA; goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa FluorTM 633, cat. no. A-21070, Invitrogen, Waltham, MA, USA) and respective isotype controls according to the manufacturer’s protocol.

Techniques: Incubation, Labeling, Confocal Microscopy, Western Blot, Immunoprecipitation

Journal: International journal of molecular sciences

Article Title: Biodistribution and Cellular Internalization of Inactivated SARS-CoV-2 in Wild-Type Mice.

doi: 10.3390/ijms23147609

Figure Lengend Snippet: Figure 8. Colocalization of SDC4 with SARS-CoV-2 in various organs of the mice. Liver, heart, lung, spleen, and brain samples of mice receiving i.v. administration of inactivated SARS-CoV-2. Tissue SDC4 is detected with an APC-labeled SDC4 antibody, while SARS-CoV-2 with an AF 488-labeled spike antibody. Scale bar = 20 µm. MOC ± SEM and PCC ± SEM for the overlap and colocalization of SARS-CoV-2 with SDC4 were calculated by analyzing 18 images from 3 samples of each animal.

Article Snippet: The SDC expression of the applied cell lines (K562 cells, SDC4 transfectants, L929, and RAW 264.7) was measured with flow cytometry by using APC-labeled anti-human SDC4 (R&D Systems, Minneapolis, MN, USA; SDC4: monoclonal rat IgG2a clone #336304, cat. no. FAB29181A) or primary anti-murine SDC antibodies (mouse SDC1, cat. no. AF3190, R&D Systems, Minneapolis, MN, USA; mouse SDC2, cat. no. PA5-95938, Invitrogen, Waltham, MA, USA; mouse SDC3, cat. no. PA5-47377, Invitrogen, Waltham, MA, USA; mouse SDC4-APC labeled antibody, cat. no. 130-109-831, Miltenyi Biotec, Bergisch Gladbach, Germany) and appropriate secondary antibodies (rabbit anti-goat IgG (H+L) cross-adsorbed secondary antibody, Alexa FluorTM 633, cat. no. A-21086, Invitrogen, Waltham, MA, USA; goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa FluorTM 633, cat. no. A-21070, Invitrogen, Waltham, MA, USA) and respective isotype controls according to the manufacturer’s protocol.

Techniques: Labeling