Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: RT-PCR of human and murine megakaryocytes and platelets. (A) Murine RT-PCR studies of total RNA from (1) megakaryocyte, (2) platelets, (3) WBCs, (4) NIH-3T3 cells, known to express LRP1,20 and (5) water for LRP1 (first gel), PF4 (second gel), and ITGB2 (third gel). Expected band size is approximately 400 base pair (bp) for LRP1 and ITGB2, and approximately 300 bp for PF4. White space indicates where lanes were removed for ease of presentation. (B) Human RT-PCR studies of total RNA from (6) megakaryocytes, (7) platelets, (8) WBCs, (9) 293T cells, embryonic kidney line known to express LRP1,21 (10) water, and (11) no reverse transcriptase controls for LRP1 (first gel), PF4 (second gel), and ITGB2 (third gel).

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: Flow cytometry and Western blot of human and murine megakaryocytes and platelets. (A) Representative examples of flow cytometry of murine bone marrow–derived megakaryocytes (top) stained with a biotin labeled anti-hLRP1 antibody known to cross-react with mouse LRP131 and then stained with streptavidin, PE–Alexa 647 secondary antibody. The gray line represents unstained cells. The broken black line represents secondary antibody alone. The solid black line is megakaryocytes with both antibodies. The bottom graph shows flow cytometry of platelets similarly performed. (B) As in panel A but for human cultured megakaryocytes and human peripheral blood platelets. LRP1 antibody was directly labeled with Alexa 647 for these experiments. As in panel A, the solid gray line represents unstained cells. The broken black line represents cells with isotype control antibody. The solid black line represents cells stained with the LRP1 antibody. (C) Western blot for LRP1 and actin as a control for protein loading. (1) Megakaryocytes, (2) platelets, and (3) WBCs. LRP1 band is expected at approximately 85 kDa and actin at approximately 25 kDa.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: Flow Cytometry, Western Blot, Derivative Assay, Staining, Labeling, Cell Culture

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: In vitro studies of the effect of RAP and anti-LRP1 antibodies on megakaryopoiesis. (A) The effect of RAP on megakaryocyte colony formation. GST indicates empty GST without conjugated RAP. Graphed is the mean percentage of megakaryocytes per well plus 1 SD. Number of experiments, each performed in duplicate, is indicated in each bar. *P = .004 versus WT cultures without PF4; **P < .003 compared with WT culture with PF4. (B) The effect of anti-LRP1 antibody (MA5A6). Ig is isoimmune control for the anti-LRP1 antibody. Mean percentage of megakaryocytes per well plus 1 SD is graphed. Number of experiments done in duplicate is indicated in each bar. *P = .004 compared with WT without PF4; **P < .003 compared with WT with PF4; ***P = .04 compared with WT without PF4.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: In Vitro

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: shRNA suppression of LRP1 and megakaryocyte colony formation. (A) Representative flow cytometry of 3T3 cells (positive control for LRP1) stably transfected with different shRNA viral vectors. The solid gray line represents cells that were unstained. The broken gray line is LRP1 expression on cells expressing the empty lentiviral vector. Solid black line shows the decrease in surface LRP1 expression after stable transfection with the LRP1 shRNA virus. (B) Same as panel A except for murine bone marrow cells after culture in media containing TPO and puromycin for 5 days. The solid gray line represents isotype control. The broken gray line is cells transfected with the negative viral vector. The solid dark line represents cells transfected with LRP1 shRNA. (C) Quantitation of change in mean fluorescence index (MFI) in murine bone marrow cells transfected with virus. Data represent mean +1 SD for 3 independent experiments. Viral titers were between 1 to 2 × 1011 viral particles/mL. (D) Effect of LRP1 shRNA on megakaryopoiesis (meg) using mPF4−/− bone marrow and hPF4High bone marrow expressed relative to megakaryopoiesis with the control empty vector. ■ is relative level of megakaryocyte seen after transfection with the empty lentiviral vector, and □ is relative level after transfection with the LRP1 shRNA vector. Data represent mean +1 SD for 4 experiments, each performed in duplicate. *P < .006 for LRP1 versus negative control for hPF4. shRNA had no effect on colony formation in mPF4−/− bone marrow. (E) Effect of LRP1 shRNA megakaryocyte colony formation in mPF4−/− bone marrow treated with exogenous PF4 (25 μg/mL). Percentage of meg colonies were normalized as in panel D. Ctl indicates control studies with empty vector. Data represent mean +1 SD. Numbers in bars represent times experiments were performed (each in duplicate). *P < .008 for LRP1 versus empty virus in the presence of PF4.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: shRNA, Flow Cytometry, Positive Control, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Quantitation Assay, Fluorescence, Negative Control

Journal: Blood

Article Title: Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes

doi: 10.1182/blood-2009-04-216473

Figure Lengend Snippet: Effect of PF4 on G1ME cells and expression of LRP1. (A) The effect of PF4 on percentage of CD42+ cells after re-expression of GATA-1 by the introduction of a GATA-1-IRES-eGFP MIGR1 retrovirus with and without 25 μg/mL PF4 in serum-free media. Results are for eGFP+-transfected cells. On the left are total CD42+ cells; middle are small, CD42+ cells; and right are large, CD42+ cells (based on forward scatter on flow cytometry). Data are shown as mean +1 SD of 4 experiments. *P < .008 comparing with and without PF4 added. (B) LRP1 expression in G1ME cells both before and after transfection with either a MIGR1 empty retrovirus (□) or MIGR1 retrovirus containing GATA-1 (♦). Figure organized as in panel A. Shown is a representative experiment of 3. (C) LRP1 expression on human cultured megakaryocytes derived from adult CD34+ bone marrow cells. Open triangles show total CD41+ cells, whereas closed triangles show LRP1+/CD41+ cells. Mean +1 SD is shown for 4 independent experiments. (D) Ploidy analysis in relation to LRP1 expression. The gray line represents cells that are CD41−. The broken line is cells that are CD41+ but LRP1−. The solid, dark line represents the cells that are positive for both LRP1 and CD41. Data are from a single experiment, but are representative of results from 5 independent experiments.

Article Snippet: In addition, in some studies, receptor-associated protein (RAP) containing low endotoxin (Molecular Innovations; 0.02 μM) or anti-LRP1 light chain antibody (MA5A6; Molecular Innovations) or anti-LRP1 heavy chain antibody (Molecular Innovations) or an anti–cluster II LRP1 antibody (R&D Systems; each at 25 μg/mL, final concentration) was added.

Techniques: Expressing, Transfection, Flow Cytometry, Cell Culture, Derivative Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation

doi: 10.1073/pnas.1909414116

Figure Lengend Snippet: M2 differentially blocks recognition of B7.1 and B7.2 by antibodies and the CD28 and CTLA4 receptors. (A, Left) MEFs transduced to express mouse B7.1 or B7.2 (MEF-mB7.1 or MEF-mB7.2) were preincubated with M2 at the indicated concentration before staining with 1 μg/mL anti-mouse B7.1 (16-10A1) or B7.2 (GL1) blocking antibody. (A, Right) Quantification of mean fluorescence intensity (MFI) of specific antibody staining in the presence of M2 relative to no M2 from 2 independent experiments is shown in the bar chart (mean ± SEM). ns, not significant. (B) Cells used in A were incubated with recombinant M2 or C8 (control PIE) and mouse CD28-Fc or CTLA4-Fc at the indicated concentration for 30 min before CD28-Fc/CTLA4-Fc binding was visualized by fluorescence-labeled anti-human IgG. (Upper) Representative flow cytometric plots of 3 independent analyses are shown. (Lower) Bar chart is the quantification of 3 analyses (mean ± SEM) showing MFI of CD28/CTLA4 in the presence of M2 relative to no M2. (C) Same experiment as in B was conducted with MEF-hB7.1 or MEF-hB7.2 and soluble human CD28-Fc or CTLA4-Fc. *P < 0.05, ***P < 0.001, ****P < 0001.

Article Snippet: The recombinant mouse and human CD28, CTLA4, and PD-L1, all fused with Fc of human IgG1 at the C terminus (except human PD-L1, fused with Fc of mouse IgG1) were from Sino Biological, Inc.

Techniques: Concentration Assay, Staining, Blocking Assay, Fluorescence, Incubation, Recombinant, Binding Assay, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation

doi: 10.1073/pnas.1909414116

Figure Lengend Snippet: M2 inhibits ex vivo T cell proliferation and activation costimulated by B7.1 and B7.2. (A) CFSE-labeled splenic T cells from C57BL/6 mice were stimulated for 3 d with MEF-mB7.2 cells in medium containing 400 nM M2, 1 μg/mL anti-mB7.2 (GL1), or IgG control in an anti-CD3ε–coated plate. CD4 cell proliferation was determined by CFSE dilution in the CD3+CD4+ population. (B) Enriched splenic T cells were cocultured with MEF-mB7.1/mB7.2 cells for 2 d in an anti-CD3ε (a-CD3)–coated plate. M2 and anti-mB7.2 antibodies were used as in A, irrelevant CPXV-PIE protein C8 and mouse CTLA4-Fc were used at 400 nM and 50 nM, respectively. The relative IL-2 production percentage in the presence of M2 or controls was calculated using splenic T cells + MEF only (+medium) as 100%. Results representative of 2 independent analyses are shown (triplicates, mean ± SD). ns, not significant; w/o, without. (C) CH27 cells were used as a stimulator in T cell activation as described in B. (D) Culture supernatants harvested at the indicated time from Vero cells infected with WT or ΔM2 CPXV (MOI = 1) were used in a T cell activation assay. Findings representative of 2 experiments (triplicates, mean ± SD) are shown. **P < 0.01, ***P < 0.001, ****P < 0001.

Article Snippet: The recombinant mouse and human CD28, CTLA4, and PD-L1, all fused with Fc of human IgG1 at the C terminus (except human PD-L1, fused with Fc of mouse IgG1) were from Sino Biological, Inc.

Techniques: Ex Vivo, Activation Assay, Labeling, Infection

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Additive Protection against Congenital Cytomegalovirus Conferred by Combined Glycoprotein B/pp65 Vaccination Using a Lymphocytic Choriomeningitis Virus Vector

doi: 10.1128/CVI.00300-16

Figure Lengend Snippet: Schematic drawing of rLCMV vector design and production. (A) LCMV vector particle and encapsidated genomic segments (GP, surface glycoprotein; NP, nucleoprotein; Z, ring finger protein; L, polymerase) and genetic organization of the two encapsidated genomic segments (S, short; L, long). (B) Representation of the full-length HCMV gB open reading frame HgB(FL), the truncated isoform HgB(dCt), and the corresponding design for guinea pig CMV gB [GPgB(dCt)] expressed in vaccine vectors. Numbers represent amino acid positions, and shaded boxes represent the proposed transmembrane region of the protein. Position 773 in HgB(dCt) is an extraneous arginine residue. (C) Schematic drawing of rLCMV vaccine vector rescue, stock production, and the single-round infectious character of rLCMV-vectored vaccination.

Article Snippet: Antibody responses against HCMV gB in mouse sera were measured by indirect ELISA using recombinant gB (Sino Biological) as coating antigen on 96-well microplates (0.5 μg/ml, 100 μl per well, overnight at 4°C) and a goat HRP-conjugated anti-mouse IgG polyclonal secondary antibody (Jackson ImmunoResearch).

Techniques: Plasmid Preparation

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Additive Protection against Congenital Cytomegalovirus Conferred by Combined Glycoprotein B/pp65 Vaccination Using a Lymphocytic Choriomeningitis Virus Vector

doi: 10.1128/CVI.00300-16

Figure Lengend Snippet: rLCMV vector characterization. (A) Growth kinetics of rLCMV vectors and LCMV wild-type virus in the production cell line 293-GP. Titers represent means of 2 independent experiments, with standard deviations shown as error bars. (B) rLCMV-gB(dCt) was selected as the representative vector to demonstrate deficiency in formation of infectious progeny in cells that do not provide LCMV GP protein in trans (293F). All cell types were infected with 0.001 FFU/cell. Samples for individual time points were analyzed by means of a FFU assay based on LCMV GP-complementing 293T cells. Dotted lines indicate detection limits of the FFU assay. (C) gB(dCt) and pp65 vaccine antigen expression levels were analyzed by Western blotting. HCMV gB and HCMV pp65-specific MAbs were used to detect vaccine antigen expression, and a polyclonal anti-LCMV serum reactive with LCMV NP was used for the infection/loading control.

Article Snippet: Antibody responses against HCMV gB in mouse sera were measured by indirect ELISA using recombinant gB (Sino Biological) as coating antigen on 96-well microplates (0.5 μg/ml, 100 μl per well, overnight at 4°C) and a goat HRP-conjugated anti-mouse IgG polyclonal secondary antibody (Jackson ImmunoResearch).

Techniques: Plasmid Preparation, Infection, Expressing, Western Blot

Journal: Virus Research

Article Title: Single amino acid substitution at position 614 in SARS-CoV-2 Spike Protein alters viral assembly and infectivity

doi: 10.1016/j.virusres.2025.199624

Figure Lengend Snippet: Effect of amino acid mutation at position 614 of S protein on viral packaging. (A and B) Western blot analysis of the protein expression of pseudovirus S protein and p24 in packaging cells and virus supernatant. (C) Schematic outline of the SARS-CoV-2 VLP production process. Plasmids encoding the SARS-CoV-2 structural proteins E, M, and N were cotransfected into HEK293T cells with plasmids encoding WT S or its mutants to obtain VLPs. (D and E) Western blot analysis of the expression of the major structural proteins S and NP on the VLPs. The original image is Supplemental Figure 4 in the supplementary material.

Article Snippet: GoldBand Plus 3-color Regular Range Protein Marker(8–180 kDa) (YEASEN, #20350ES72); SARS-CoV-2 (2019-nCoV) Spike RBD Antibody, Rabbit PAb, Antigen Affinity Purified (Sinobiological, # 40,592-T62); SARS-CoV-2 (2019-nCoV) Spike S2 Antibody, Rabbit PAb, Antigen Affinity Purified (Sinobiological, #40,590-T62); GFP (4B10) Mouse mAb (CST, #2955); Flag (DYKDDDDK) tag Polyclonal Antibody (Proteintech, #20,543–1-AP); Human Immunodeficiency Virus type 1 (HIV-1) Gag-p24 Antibody, Rabbit PAb, Antigen Affinity Purified (Sinobiological, #11,695-RP02); Myc-Tag (9B11) Mouse mAb (CST, #2276); GAPDH Mouse Monoclonal Antibody (Origene, #OTI2D9); β-actin Polyclonal Antibody (Proteintech, #20,536–1-AP).

Techniques: Mutagenesis, Western Blot, Expressing, Virus