rabbit anti human cd47  (Proteintech)

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a Overview of the outline for establishing H1975 and HEK293T cell lines expressing a fusion GFP gene in the C-terminus of CD47 (CD47-GFP) and subsequently identifying the deubiquitinase of CD47 through DUB siRNA library screening with two stable cell lines. DUBs deubiquitinases; FACS fluorescence-activated cell sorting; MFI mean fluorescence intensity. b Whole-cell lysates (WCL) of H1975 or PC9 cells treated with indicated deubiquitinase inhibitors (2.5 µM) or dimethyl sulfoxide (DMSO) for 12 hours (h) were prepared and subjected to immunoblotting (IB) analysis. c – e IB analysis of WCL derived from H1975 cells ( c ) and PC9 cells ( e ) treated with ML364 (1 µM and 2 µM) or DMSO for 16 h. The mRNA level of CD47 in H1975 cells ( c ) was measured using reverse transcription quantitative PCR (RT-qPCR) ( d ). f , g Immunofluorescence (IF) staining for CD47 in H1975 cells ( f ) and PC9 cells ( g ) treated with ML364 (2 µM) for 16 h. Scale bar, 25 μm. h – k IB analysis of WCL derived from H1975 ( h ) or PC9 ( j ) cells stably expressing sh USP2 or sh GFP , respectively. The mRNA level of CD47 in H1975 ( i ) or PC9 ( k ) cells was measured using RT-qPCR. l , m IB analysis of Cd47 protein expression in the tissues of lung, heart, or kidney obtained from wild-type (WT) and Usp2 −/− mice ( l ). Quantification of Cd47 protein band intensity was normalized to vinculin ( m ). n – p IB analysis of WCL derived from HEK293T cells co-transfected with indicated constructs ( n , p ). The mRNA level of CD47 was quantified by RT-qPCR ( o ). EV: empty vector. q , r IB analysis of WCL derived from HEK293T cells co-transfected with indicated constructs. Cells were treated with 200 μg/ml CHX for the indicated time points ( q ). Quantification of CD47 protein band intensity was normalized to vinculin, then compared to the t = 0 time point ( r ). s , t Representative images from immunohistochemical (IHC) staining of CD47 and USP2 in human lung adenocarcinoma ( s ). Scale bar, left panels: 100 μm; right panels: 50 μm. n = 83. Quantification of USP2 and CD47 staining intensities was performed as average optical density (AOD) [AOD = Integrated Optical Density (IOD) SUM/Area SUM] (t) . For ( d , i , k , m , o , and r ), unpaired two-tailed Student’s t -test. Correlations were analyzed by Pearson’s test ( t ). Data are shown as the mean ± SD, n = 3 independent biological replicates. P < 0.05 was considered statistically significant. n = 3 biologically independent experiments for ( b , c , e , h , j , l , n , p , and q ). Source data are provided as the Source Data file.

Article Snippet: Anti-human CD47 (D3O7P) Rabbit mAb (63000, 1:200), anti-mouse CD8α Rabbit mAb (98941, 1:200), anti-human CD8 Rabbit pAb (85336, 1:200), and anti-human Pan-Keratin (C11) Mouse mAb (4545, 1:400) were purchased from Cell Signaling Technology.

Techniques: Expressing, Library Screening, Stable Transfection, Fluorescence, FACS, Western Blot, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Staining, Transfection, Construct, Plasmid Preparation, Immunohistochemical staining, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a IB analysis of HEK293T WCL and anti-HA immunoprecipitates (IPs). HEK293T cells were co-transfected with indicated constructs and treated with 10 µM MG132 for 12 h before harvesting. b IB analysis of glutathione S-transferase (GST) pull-down protein mixture from HEK293T cell lysates that overexpressed CD47-cHA incubated with bacterially purified recombinant GST or GST-USP2 protein. c IB analysis of GST pull-down products from HEK293T cell lysates that overexpressed Flag-USP2 incubated with bacterially purified recombinant GST or GST-CD47 protein. d Schematic representation of WT and truncations of USP2, including the N-terminal region of amino acid (aa) 1–266 and C-terminal domain of aa267–605. e IB analysis of WCL and anti-HA IPs obtained from HEK293T cells, which were co-transfected with indicated constructs and treated with 10 µM MG132 for 12 h before harvesting. f IB analysis of GST pull-down products derived from HEK293T cell lysates that ectopic expression of CD47-cHA incubated with bacterially purified recombinant GST, GST-USP2 WT, and GST-USP2 truncations. g IB analysis of GST pull-down products derived from HEK293T cell lysates that ectopic expression of Flag-USP2 WT and truncations incubated with bacterially purified recombinant GST-CD47 protein. h Schematic diagram of CD47 WT and its various deletion mutants. i IB analysis of GST pull-down products derived from HEK293T cell lysates that ectopic expression of CD47-cHA WT and deletion mutants incubated with bacterially purified recombinant GST-USP2 protein. j , k IB analysis of WCL and IPs derived from H1975 ( j ) and PC9 ( k ) cells. l , m IB analysis of WCL and Ni-NTA pull-down products of the in vivo ubiquitination assay in the guanidine-HCl denaturing buffer. HEK293T cells were co-transfected with the indicated constructs and treated with 10 µM MG132 for 12 h before harvesting. n IB analysis of WCL and Ni-NTA pull-down products of the in vivo ubiquitination assay in the guanidine-HCl denaturing buffer. HEK293T cells were co-transfected with the indicated constructs and treated with 2 µM ML364 for 16 h and 10 µM MG132 for 12 h before harvesting. o , p IB analysis of WCL and IPs derived from lysates of H1975 ( o ) and PC9 ( p ) cells using indicated K48-Ubi antibodies. Cells were treated with 20 µM MG132 for 6 h before harvesting. q , r IB analysis of WCL and anti-CD47 IPs derived from H1975 ( q ) or PC9 ( r ) cells stably expressing sh USP2 or sh GFP , respectively. Cells were treated with 20 µM MG132 for 6 h before harvesting. n = 3 biologically independent experiments for ( a , b , c , e , f , g , and i – r ). Source data are provided as the Source Data file.

Article Snippet: Anti-human CD47 (D3O7P) Rabbit mAb (63000, 1:200), anti-mouse CD8α Rabbit mAb (98941, 1:200), anti-human CD8 Rabbit pAb (85336, 1:200), and anti-human Pan-Keratin (C11) Mouse mAb (4545, 1:400) were purchased from Cell Signaling Technology.

Techniques: Transfection, Construct, Incubation, Purification, Recombinant, Derivative Assay, Expressing, In Vivo, Ubiquitin Proteomics, Stable Transfection

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a A schematic treatment plan for immunocompetent C57BL/6J mice bearing LLC tumors. Mice were subcutaneously implanted with 2 × 10 6 LLC cells and treated with a control vehicle, USP2 inhibitor (ML364, 5 mg/kg, daily for 12 doses), anti-PD-1 mAb (100 μg per mouse, every three days for 4 treatments), or combined treatment, respectively. i.p., intraperitoneal; s.c., subcutaneous; MAb, monoclonal antibody. b , c Tumor growth ( b ) or Kaplan–Meier survival curves ( c ) for C57BL/6J bearing LLC tumors with indicated treatments. d The weight of LLC tumors was measured at the endpoint. e Quantification of cellular surface CD47 on CD45 − cells in subcutaneous LLC tumors derived from C57BL/6J mice with indicated treatments. f – h Quantification of MHCII + (M1 macrophage) cells ( f ) or CD206 + (M2 macrophage) cells ( g ) represented as percentage of F4/80 + macrophages, and CD8 + T cells ( h ) represented as percentage of CD3 + T cells in subcutaneous LLC tumors derived from C57BL/6J mice-indicated treatments. i A schematic treatment plan for the immunocompetent C57BL/6J lung tumor mouse model. Mice were injected with 1 × 10 6 LLC cells/per mouse via tail vein injection, followed by treatments with control vehicle, ML364 (5 mg/kg), anti-PD-1 mAb (100 μg per mouse), or combined treatment, respectively. i.p., intraperitoneal; i.v., intravenous. j – l Representative H&E-stained images of lung tissues from mice across different groups ( j ), tumor size was quantified by measuring the cross-sectional area of all tumors ( k ), and the tumor-occupied lung area (tumor area, %) was calculated ( l ) from three nonconsecutive sections per mouse. Scale bars, 5 mm. m LLC-GFP tumors bearing C57BL/6J mice were treated as ( a ). Representative images of IF staining for tumor cells (GFP, green), macrophages (F4/80, red), and nuclei (DAPI, blue). The yellow (indicated with arrows) showing colocalization of GFP (green) and F4/80 (red) double-positive cells suggested the macrophage phagocytosis of tumor cells. Each point represents the average counts of yellow cells within three high-power fields. The sections were scanned at 60× with an oil objective on a confocal microscope. Scale bars, 50 μm (left panels); zoom scale bars, 20 μm (right panels). For ( d – h , k , and l ), unpaired two-tailed Student’s t -test. Two-way ANOVA for ( b ). Log-rank test for ( c ). n = 5 mice per group. Data are shown as the mean ± SD. P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.

Article Snippet: Anti-human CD47 (D3O7P) Rabbit mAb (63000, 1:200), anti-mouse CD8α Rabbit mAb (98941, 1:200), anti-human CD8 Rabbit pAb (85336, 1:200), and anti-human Pan-Keratin (C11) Mouse mAb (4545, 1:400) were purchased from Cell Signaling Technology.

Techniques: Control, Derivative Assay, Injection, Staining, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a Diagrammatic treatment plan for immunocompetent C57BL/6J mice with LLC tumors. Mice were implanted with 2 × 10 6 LLC cells and treated with Vehicle, ML364 (5 mg/kg daily for 18 doses), anti-PD-1 mAb (100 μg/mouse every three days for 6 doses), or combination therapy. i.p., intraperitoneal; s.c., subcutaneous. b Tumor volume in different treatment groups was assessed using calipers, and the tumor growth curve was generated. n = 8 (Vehicle); 8 (ML364); 8 (anti-PD-1 mAb) and 10 (Combined therapy) mice. c Kaplan–Meier survival curves for C57BL/6J bearing LLC tumors with indicated treatments. Log-rank test. n = 8 (Vehicle); 8 (ML364); 8 (anti-PD-1 mAb) and 10 (Combined therapy) mice. d A schematic treatment plan for immunocompetent C57BL/6J mice bearing 2 × 10 6 LLC tumors. Mice were treated with a control vehicle, USP2 inhibitor (ML364, 30 mg/kg for 18 doses), anti-PD-1 mAb (200 μg per mouse for 6 doses), or combined treatment, respectively. e Tumor volume in the different treatment groups was measured using calipers, and the corresponding tumor growth curve was generated. Vehicle (n = 9), ML364 (n = 8), PD-1 mAb (n = 8), ML364 & PD-1 mAb (n = 13) mice per group. f Kaplan–Meier survival curves for C57BL/6J bearing LLC tumors with indicated treatments. Vehicle (n = 9), ML364 (n = 8), PD-1 mAb (n = 8), ML364 & PD-1 mAb (n = 13) mice per group. Log-rank test. g IHC staining for CD47 and PD-L1 in LLC tumors from C57BL/6J mice treated with indicated reagents. Scale bar, 50 μm. h , i Quantification for CD47 ( h ) and PD-L1 ( i ) positive cells. Each point represents the average counts of positive cells within three high-power fields. For ( h ) and ( i ), unpaired two-tailed Student’s t -test. n = 5 mice per group. Log-rank test for ( c , f ). Data are shown as the mean ± SD. P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.

Article Snippet: Anti-human CD47 (D3O7P) Rabbit mAb (63000, 1:200), anti-mouse CD8α Rabbit mAb (98941, 1:200), anti-human CD8 Rabbit pAb (85336, 1:200), and anti-human Pan-Keratin (C11) Mouse mAb (4545, 1:400) were purchased from Cell Signaling Technology.

Techniques: Generated, Control, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a A schematic treatment plan for immunocompetent C57BL/6J mice bearing sg Control- or sg Cd47- LLC tumors. Mice were subcutaneously implanted with 2 × 10 6 sg Control or sg Cd47 -LLC cells and treated with a control vehicle, USP2 inhibitor (ML364, 5 mg/kg, daily for 12 doses), anti-PD-1 mAb (100 μg per mouse, every three days for 4 treatments), or combined treatment, respectively. i.p., intraperitoneal; s.c., subcutaneous. b , c Tumor volume in each treatment group was measured using calipers, and the tumor growth curve was subsequently plotted ( b ). At the study endpoint, the weight of LLC tumors was recorded ( c ). n = 5 mice per group. d The body weight of C57BL/6J mice was monitored throughout the duration of the indicated treatment. n = 5 mice per group. e A schematic treatment plan for CD8 T cell or macrophage deletion experiments. Mice were randomly divided into five groups: (1) vehicle, (2) ML364 & PD-1 mAb (MP), (3) MP & CD8 deletion (αCD8), (4) MP & macrophage deletion (clodronate liposomes, CL), and (5) MP & αCD8 & CL. On day 5 post-tumor implantation, mice in MP were treated with a combination of ML364 (5 mg/kg) daily for 12 treatments and anti-PD-1 mAb (100 μg per mouse) every 3 days for 4 doses. After grouping as indicated, mice received αCD8 antibody (200 μg per mouse) every 3 days for 4 doses, starting three days before tumor implantation. Macrophage deletion by CL was administered with 200 μl per mouse every 3 days for 4 doses, starting on the day of tumor implantation. i.p., intraperitoneal; s.c., subcutaneous. f , g Tumor volume for each treatment group was measured using calipers, and the tumor growth curve was plotted ( f ). The weight of LLC tumors was measured at the endpoint ( g ). n = 5 mice per group. h , i Quantification of CD8 + represented as percentage of CD3 + T cells ( h ) and F4/80 + macrophages represented as percentage of CD11b + cells ( i ) in subcutaneous LLC tumors derived from C57BL/6J mice treated with indicated reagents. For ( c ) and ( g – i ), unpaired two-tailed Student’s t -test. n = 5 mice per group. Two-way ANOVA for ( b , d , and f ). Data are shown as the mean ± SD. P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.

Article Snippet: Anti-human CD47 (D3O7P) Rabbit mAb (63000, 1:200), anti-mouse CD8α Rabbit mAb (98941, 1:200), anti-human CD8 Rabbit pAb (85336, 1:200), and anti-human Pan-Keratin (C11) Mouse mAb (4545, 1:400) were purchased from Cell Signaling Technology.

Techniques: Control, Liposomes, Tumor Implantation, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: USP2 inhibition unleashes CD47-restrained phagocytosis and enhances anti-tumor immunity

doi: 10.1038/s41467-025-59621-5

Figure Lengend Snippet: a , b Representative IF staining images for CD163 (green) and CD8 (red) in human lung adenocarcinoma (LUAD) tissues with the USP2 high (n = 13) and low (n = 16) expression ( a ). Quantifications of CD163 and CD8 positive cells in LUAD samples with USP2 high or low expression, respectively ( b ). Each point represents the average counts of positive cells within three high-power fields. Scale bars, 500 μm (left panels); zoom scale bars, 50 μm (right panels). c – e USP2 expression was negatively correlated with the infiltration of M1 macrophages ( c ) and CD8 + T cells ( e ), while USP2 displayed a positive correlation with the infiltration of M2 macrophages ( d ) in lung cancer patients based on the immune association analysis by TIMER2.0 software. f , g Representative images from IHC staining of CD47 and USP2 in responders (R) and non-responders (NR) of lung cancer patients with anti-PD-1 therapy ( f ). Scale bar, left panels: 100 μm; right panels: 50 μm. Association between USP2 or CD47 expression and response to anti-PD-1 therapy in lung cancer patients ( g ). n = 6 (R), or 6 (NR). Patients were divided into USP2 or CD47 low and high expression groups according to their IHC score mean value ( f ). h , i Representative images of multiplex immunohistochemistry (mIHC) staining for R and NR of lung cancer patients with anti-PD-1 immunotherapy ( h ). Quantifications of CD163 (white), CD8 (red), and CK (green) in each group ( i ). Each point represents the average counts of positive cells within three high-power fields. n = 5 per group. Scale bars, 100 μm (left panels); zoom scale bars, 50 μm (right panels). j , k Representative images from IHC staining of CD47 and USP2 in responders and non-responders of oral cancer patients with anti-PD-1 therapy ( j ). Scale bar, left panels: 100 μm; right panels: 50 μm. Association between USP2 or CD47 expression and response to anti-PD-1 therapy in oral cancer patients ( k ). n = 25 (R), or 19 (NR). Patients were divided into USP2 or CD47 low and high expression groups according to their IHC score mean value ( j ). l , m Representative images of mIHC staining for responders and non-responders of oral cancer patients with anti-PD-1 therapy ( l ). Quantifications of CD163 (white), CD8 (red), and CK (green) in each group ( m ). Each data point represents the average counts of positive cells within three high-power fields. n = 18 (R), or 13 (NR). Scale bars, 100 μm (left panels); zoom scale bars, 50 μm (right panels). For ( b ), unpaired two-tailed Student’s t -test. For ( g ) and ( k ), a two-sided χ 2 test. For ( i ) and ( m ), two-tailed Non-parametric Mann–Whitney test. Data are shown as the mean ± SD P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.

Article Snippet: Anti-human CD47 (D3O7P) Rabbit mAb (63000, 1:200), anti-mouse CD8α Rabbit mAb (98941, 1:200), anti-human CD8 Rabbit pAb (85336, 1:200), and anti-human Pan-Keratin (C11) Mouse mAb (4545, 1:400) were purchased from Cell Signaling Technology.

Techniques: Staining, Expressing, Software, Immunohistochemistry, Multiplex Assay, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: The CIS association of CD47 with integrin Mac-1 regulates macrophage responses by stabilizing the extended integrin conformation

doi: 10.1016/j.jbc.2023.103024

Figure Lengend Snippet: Association of CD47 with Mac-1 on the surface of various Mac-1–expressing cells probed by immunoprecipitation analyses.A, purified WT and CD47-deficient inflammatory peritoneal mouse macrophages were lysed and immunoprecipitated with rabbit polyclonal anti-αM antibody, rabbit polyclonal anti-CD47 antibody, or control rabbit IgG that was used as a specificity control. Blots were analyzed with rabbit polyclonal antibodies against the αM or CD47. The lysates of WT and CD47-deficient macrophages were analyzed by Western blotting using anti-αM and CD47 antibodies. B, murine IC-21 macrophages were lysed and immunoprecipitated with anti-αM rabbit polyclonal antibody or rabbit polyclonal anti-CD47 antibody, and blots were analyzed with rabbit polyclonal antibody against the αM or CD47. C, biotinylated Mac-1-HEK293 cells were lysed and immunoprecipitated with mAb 44a against the αM subunit or isotype control IgG1. Blots were disclosed with streptavidin-conjugated horseradish peroxidase (HRP). The molecular weight of the αM (165 kDa) and β2 (95 kDa) integrin subunits and CD47 (47 kDa) are indicated on the right of the panel. D, suspended (denoted “s”) or adherent (denoted “a”) Mac-1-HEK293 cells were lysed and immunoprecipitated with anti-αM mAb 44a or anti-β2 mAb IB4. Blots were analyzed with anti-αM, anti-β2, and anti-CD47 antibodies. E, the ratios of CD47 to the αM and β2 integrin subunits in the immunoprecipitates from suspended and adherent cells were determined from the densitometry analyses of blots. The ratio of CD47 to each integrin subunit in suspended cells was taken as 1.0. F, lysates of biotinylated Mac-1-HEK293 cells were immunoprecipitated with anti-CD47 mAb B6H12; then immunoprecipitates were subjected to Western blotting probed with streptavidin-HRP (left panel; 1 IP). After the first round of immunoprecipitation, the supernatant was immunoprecipitated with anti-αM mAb 44a (middle panel; 2 IP). The third round of immunoprecipitation (3 IP) was performed using anti-β1 mAb (right panel). M, molecular weight markers.

Article Snippet: The rabbit polyclonal anti-human/mouse CD47 antibody (catalog #CD47-101AP) was from FabGennix.

Techniques: Expressing, Immunoprecipitation, Purification, Western Blot, Molecular Weight

Journal: Journal of Cell Communication and Signaling

Article Title: CD47 interactions with exportin-1 limit the targeting of m 7 G-modified RNAs to extracellular vesicles

doi: 10.1007/s12079-021-00646-y

Figure Lengend Snippet: CD47 regulates m 7 G-modified miRNAs in EVs released by Jurkat T cells and their association with exportin-1. a Analysis of known m 7 G-modified miRNAs that were differentially expressed in WT (blue) or CD47 − (red) EVs vs cells ( p < 0.05) (from Data S2C, Fig S4N,O). b Gene set enrichment analysis (GSEA) of exportin-1-dependent miRNAs in EVs released from WT versus CD47 − cells. c EVs released by WT and CD47 − cells were isolated by density gradient centrifugation for 16 h, lysed, and levels of the indicated miRNAs was analyzed. RNA–IP was performed from the same lysates using m 3 G/m 7 G-cap antibody, and miRNAs were quantified using real-time PCR and normalized to input. d–f CD47-dependent association of known m 7 G-cap-dependent pre-miRNAs with exportin-1. Association of the indicated pre-miRNAs with exportin-1 was quantified using RNA-immunoprecipitation with anti-exportin-1 from WT and CD47 − T cells followed by qPCR analysis. Enrichments were calculated after normalizing all input values to one. g–j Effects of TSP1 and inhibiting exportin-1 with LMB on miRNA maturation. miRNA levels of miR-320a-3p and pre-miR-320a (Qiagen) in untreated and TSP1 treated WT and CD47 − T cells and released EVs purified using Exo-spin™

Article Snippet: The cells were fixed and immunostained as previously described (Kaur et al. ) using rabbit anti-human CD47, mouse anti-human CD47, rabbit anti-human exportin-1 (Proteintech), human CD47 antibody B6H12, exportin-1/CRM1 from Santa Cruz Biotechnologies, RANBP1, RANGAP1 from Cell Signaling.

Techniques: Modification, Isolation, Gradient Centrifugation, Real-time Polymerase Chain Reaction, RNA Immunoprecipitation, Purification

Journal: Journal of Cell Communication and Signaling

Article Title: CD47 interactions with exportin-1 limit the targeting of m 7 G-modified RNAs to extracellular vesicles

doi: 10.1007/s12079-021-00646-y

Figure Lengend Snippet: CD47 differentially regulates the abundance of specific miRNAs in cells and EVs. a Hierarchical clustering of differentially expressed miRNAs in EVs (> twofold, p val < 0.05) with unsupervised clustering of their parental cell data based on microarray analyses of WT and CD47 deficient Jurkat T cells and their released EVs (N = 3) b Hierarchical clustering of differentially expressed cellular miRNAs with unsupervised EV data . c Principal component analysis of miRNA data for WT and CD47 − Jurkat T cells and EVs released by these cells. d Venn diagram summarizing RNAseq analysis of differentially expressed miRNAs ( p < 0.05) comparing WT and CD47 − cells (Blue) and EVs (Red) (Data S2A, p < 0.05). The left pie chart depicts the percentages of miRNAs up- or down-regulated only in CD47 − versus WT EVs. The right pie chart presents the percentages of miRNAs up (orange) or down regulated (gray) in CD47 − EVs and in cells and miRNAs showing opposite CD47-dependent regulation in cells versus EVs (dark blue). e, f Venn diagram presenting numbers of precursor e and mature miRNAs f differentially expressed in WT vs. CD47 − cells (blue) and WT vs CD47 − EVs (red) (Data S2A, p < 0.05). g Confirmation using real time PCR of representative differentially regulated miRNAs between WT and CD47 − cells and their EVs (Data S1C). h WT cells were transfected with a CD47 guide CRISPR/Cas9and grown in complete medium for 24 h. Transfected cells were transferred into HITES serum free medium for 24 h. EVs were isolated using the Exo-quick Kit, and miRNA levels of miR-31, miR-107 and miR-103 was analyzed using real-time PCR. Significance is indicated for P -values ≤ 0.05 for comparisons of Sample and Columns (*) or Sample, Columns and Interaction (**) as detailed in the Statistical Analysis method section. i CD47 − cells were transfected for 48 h with control or CD47 expression plasmids. Total RNA from cells and EVs was analyzed for expression of the indicated miRNAs. The level was normalized to EVs extracted from WT using U6 as control. Significance was determined by two-sample t-test assuming equal variances ( P -value ≤ 0.05). j WT and CD47 − T Cells were transferred into serum free HITES medium for 24 h. EVs were isolated using ultracentrifugation basic protocol 1 and k size exclusion chromatography purification using Exo-guidance systems, and real time PCR was performed. l RNA was extracted from CD3 + T cells isolated from WT and Cd47 −/− mice, and miRNA level was analyzed using real time PCR. Significance is indicated for P -values ≤ 0.05 with two-sample t-test: assuming equal variances

Article Snippet: The cells were fixed and immunostained as previously described (Kaur et al. ) using rabbit anti-human CD47, mouse anti-human CD47, rabbit anti-human exportin-1 (Proteintech), human CD47 antibody B6H12, exportin-1/CRM1 from Santa Cruz Biotechnologies, RANBP1, RANGAP1 from Cell Signaling.

Techniques: Microarray, Real-time Polymerase Chain Reaction, Transfection, CRISPR, Isolation, Control, Expressing, Size-exclusion Chromatography, Purification

Journal: Journal of Cell Communication and Signaling

Article Title: CD47 interactions with exportin-1 limit the targeting of m 7 G-modified RNAs to extracellular vesicles

doi: 10.1007/s12079-021-00646-y

Figure Lengend Snippet: CD47 localization in EVs and interactions with the Ran/exportin-1 complex. a Cell lysates from WT and CD47 − Jurkat cells were immunoprecipitated with streptavidin beads using a biotin-conjugated CD47 antibody (CC2C6). Proteins eluted from the beads were digested and subjected to LC–MS analysis to identify CD47-interacting proteins. A is the number of unique peptides, those peptides with unique sequence that are only found in the given protein. B is the number of total peptides, which is those with unique sequence that are found in the given protein but may also be found in other proteins. C is the number of peptide spectral matches, which is the total number of MS/MS spectra representing peptides from the given protein, including redundant spectra for the same peptide sequence. b–d The interactions identified by LC–MS were validated using immunoprecipitation of CD47, ubiqulin-1 and exportin-1. e Representative blot showing effects of LMB and GTPγS treatment on immunoprecipitation of exportin-1 with ubiquilin-1 in Jurkat whole cell lysates f Quantitative analysis of 3 replicate experiments with exportin-1 protein density normalized to ubiquilin-1. g, h Representative exportin-1 immunoblot and quantification of three replicate experiments (H) showing effects of LMB and GTPγS treatment on the association of exportin-1 with CD47 immunoprecipitated using CC2C6, p = .033 (*), i–l Electron micrographs for immunogold labeling to localize CD47 in vehicle treated i, k and LMB treated (J.L) Jurkat T cells. Open arrowheads indicate CD47 in the plasma membrane. Boxed areas in I and J, enlarged in K and L, respectively, show intracellular labeling for CD47 localized to MVBs. Scale bar for I-J indicates 1 μm; scale bars for K-L indicate 200 nm. m CD47 immunogold labeled and negative stained electron micrograph of EVs released by WT T cells and isolated using Exo-spin. Boxed area around one EV is shown enlarged to the right (top), with immunogold particles circled in white below. Scale bars indicate 100 nm (left), and 50 nm (right). n Immunoprecipitation of exportin-1 was performed using lysates (± Triton-X100) of EVs derived from WT and CD47 − cells or cells treated with 20 nM LMB. Western blotting was performed to detect CD47 (glycosylated isoforms indicated by arrows) and exportin-1

Article Snippet: The cells were fixed and immunostained as previously described (Kaur et al. ) using rabbit anti-human CD47, mouse anti-human CD47, rabbit anti-human exportin-1 (Proteintech), human CD47 antibody B6H12, exportin-1/CRM1 from Santa Cruz Biotechnologies, RANBP1, RANGAP1 from Cell Signaling.

Techniques: Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Sequencing, Tandem Mass Spectroscopy, Western Blot, Labeling, Clinical Proteomics, Membrane, Staining, Isolation, Derivative Assay

Journal: Journal of Cell Communication and Signaling

Article Title: CD47 interactions with exportin-1 limit the targeting of m 7 G-modified RNAs to extracellular vesicles

doi: 10.1007/s12079-021-00646-y

Figure Lengend Snippet: Known RanGTP-dependent exportin-1 binding proteins that associate specifically with CD47 in T cells

Article Snippet: The cells were fixed and immunostained as previously described (Kaur et al. ) using rabbit anti-human CD47, mouse anti-human CD47, rabbit anti-human exportin-1 (Proteintech), human CD47 antibody B6H12, exportin-1/CRM1 from Santa Cruz Biotechnologies, RANBP1, RANGAP1 from Cell Signaling.

Techniques: Binding Assay, Ubiquitin Proteomics, Activity Assay

Journal: Journal of Cell Communication and Signaling

Article Title: CD47 interactions with exportin-1 limit the targeting of m 7 G-modified RNAs to extracellular vesicles

doi: 10.1007/s12079-021-00646-y

Figure Lengend Snippet: CD47 and TSP1 regulation of m 7 G-cap-dependent mRNA trafficking in human and murine T cells. a GSEA analysis shows enrichment of EIF4E-dependent RNAs in EVs released from CD47 − versus WT Jurkat T cells. b Venn Diagram representing overlap between CD47-dependent mRNAs in cells and EVs with previously reported EIF4E-dependent capped RNAs (Culjkovic-Kraljacic et al. ). c, d Expression of YOD1, EEFIA1 and ETS1 mRNAs in untreated WT and CD47 − Jurkat T cells and cells treated with TSP1 or LMB. EVs were isolated using Exo-spin™ technology. e Expression of Yod1, Eefia1 and Ets1 in control, and LMB or TSP1 treated WT and Cd47 −/− mouse T cells using total cellular RNA. f – j Cytosolic and nuclear mRNA expression of YOD1, EEFIA1, CUL7, ETS1 and XPO1 in untreated WT and CD47 − Jurkat T cells and cells treated with 1 μg/ml TSP1. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)

Article Snippet: The cells were fixed and immunostained as previously described (Kaur et al. ) using rabbit anti-human CD47, mouse anti-human CD47, rabbit anti-human exportin-1 (Proteintech), human CD47 antibody B6H12, exportin-1/CRM1 from Santa Cruz Biotechnologies, RANBP1, RANGAP1 from Cell Signaling.

Techniques: Expressing, Isolation, Control

Journal: Journal of Cell Communication and Signaling

Article Title: CD47 interactions with exportin-1 limit the targeting of m 7 G-modified RNAs to extracellular vesicles

doi: 10.1007/s12079-021-00646-y

Figure Lengend Snippet: TSP1 regulates the subcellular localization of exportin-1 and RanBP1 in a CD47-dependent manner. a, b Confocal microscopic analysis of CD47/exportin-1 and exportin-1/RanBP1 in T cells treated with LMB and TSP1 (1 μg/ml) for 1 h. Scale bar = 5 μm. c Western blots with anti-exportin-1 and RanBP1 in cytosolic, nuclear, and EV fractions from untreated WT and CD47 − T cells and cells treated with LMB and TSP1. d – g Quantification of western blots by calculating the ratio of nuclear/cytosolic was determined by normalizing to immunoblots for GAPDH or CROP loading controls, respectively (n = 2)

Article Snippet: The cells were fixed and immunostained as previously described (Kaur et al. ) using rabbit anti-human CD47, mouse anti-human CD47, rabbit anti-human exportin-1 (Proteintech), human CD47 antibody B6H12, exportin-1/CRM1 from Santa Cruz Biotechnologies, RANBP1, RANGAP1 from Cell Signaling.

Techniques: Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: CD47 interactions with exportin-1 limit the targeting of m 7 G-modified RNAs to extracellular vesicles

doi: 10.1007/s12079-021-00646-y

Figure Lengend Snippet: CD47/ubiquilin-1 interact with and regulate the Ran/exportin-1 nuclear transport complex, which mediates trafficking of m 7 G-modified RNAs to the cytoplasm and extracellular vesicles (EVs). CD47 signaling regulates nuclear/cytoplasmic transport of m 7 G-capped RNAs and their abundance in EVs. Leptomycin B (LMB) inactivates exportin-1 (XPO1) by covalent modification, which inhibits the export of cargo proteins and m 7 G-capped RNAs from the nucleus. LMB also inhibits the association of exportin-1 with CD47 and enhances levels of m 7 G-capped RNAs in EVs released from cells. The mechanism by which exportin-1 and CD47 regulate RNAs destined for direct EV release versus release via multivesicular bodies (MVBs) remains to be determined

Article Snippet: The cells were fixed and immunostained as previously described (Kaur et al. ) using rabbit anti-human CD47, mouse anti-human CD47, rabbit anti-human exportin-1 (Proteintech), human CD47 antibody B6H12, exportin-1/CRM1 from Santa Cruz Biotechnologies, RANBP1, RANGAP1 from Cell Signaling.

Techniques: Modification