Journal: eLife

Article Title: Phenotypic analysis of the unstimulated in vivo HIV CD4 T cell reservoir

doi: 10.7554/eLife.60933

Figure Lengend Snippet:

Article Snippet: Antibody , CD49d (α4) (mouse monoclonal) , Fluidigm , Cat#3141004B , (1 μg/100 μL).

Techniques:

Journal: eLife

Article Title: Phenotypic analysis of the unstimulated in vivo HIV CD4 T cell reservoir

doi: 10.7554/eLife.60933

Figure Lengend Snippet:

Article Snippet: Antibody , CD49d (α4) (mouse monoclonal) , Fluidigm , Cat#3141004B , (1 μg/100 μL).

Techniques:

Journal: Communications Biology

Article Title: BLM and FANCJ role in the response to G-quadruplex-dependent telomeric replicative stress

doi: 10.1038/s42003-025-09367-z

Figure Lengend Snippet: a The diagram shows the workflow on a timeline: on the day after cell seeding, cells were treated with RHPS4 (0.2 and/or 0.5 μM). At 48 h after treatment, cells were again treated with RHPS4 (0.2 and/or 0.5 μM) or harvested and counted. At 72 h after the first treatment, the cell viability was checked, and finally at 96 h after the first treatment, the cells were harvested and processed. b Representative images showing U251MG cells stained by immunofluorescence using anti-BLM (red signals), and anti-TRF1 or anti-TRF2 (green signals) antibodies. Merged images allow visualization of colocalizing dots (yellow signals). Yellow arrows indicate BLM and TRF1-TRF2 colocalizations. c Quantification of the colocalizations between BLM and TRF1 or TRF2 proteins in U251MG cell line upon RHPS4 treatment (0.2 and 0.5 μM). d , e Sensitivity of U251MG and BLM −/− cell lines to RHPS4 concentrations ranging from 0.01 to 2 μM (0.01; 0.125; 0.25; 0.5; 1; 2 μM), evaluated 96 h after the first treatment. Mitomycin C (MMC) was used as a positive control at concentrations of 0.1; 0.5; 1; 2; 5 μg/ml. The Sulforhodamine B (SRB) cytotoxicity assay showed that RHPS4 sensitivity was unchanged in U251MG and BLM −/− (IC50 was 0.56 μM in both cell lines). f Short-term cell proliferation in untreated cells, U251MG and BLM −/− , and in RHPS4-treated cells (0.5 μM) as evaluated 48 and 96 h after the first treatment. g Long-term cell proliferation in U251MG and BLM −/− untreated and RHPS4-treated cells (0.5 μM). Scale bars represent 5 μm. * p < 0.05, ** p < 0.01 (two-way ANOVA; n = 3). Error bars denote the standard deviation of the mean.

Article Snippet: The slides were then incubated in blocking buffer (1% BSA dissolved in 1X PBS [w/v]) and then incubated overnight (ON) at 4 °C with the primary antibodies (rabbit polyclonal anti-BLM (#A300-110A, Bethyl) (1:100); mouse monoclonal anti-TRF1 (4E4 clone, GTX70304, GeneTex) (1:20) or mouse monoclonal anti-TRF2 (9F10 clone, sc-47693, Santa Cruz Biotechnology) (1:100)).

Techniques: Staining, Immunofluorescence, Positive Control, Cytotoxicity Assay, Standard Deviation

Journal: Journal of Cell Science

Article Title: Multiple E2 ubiquitin-conjugating enzymes regulate human cytomegalovirus US2-mediated immunoreceptor downregulation

doi: 10.1242/jcs.206839

Figure Lengend Snippet: UBE2G2 and UBE2J2 regulate US2-induced immunoreceptor downregulation. (A,B) U937 (A) or THP-1 (B) cells expressing US2 were lentivirally transduced with a CRISPR/Cas9 vector targeting TRC8 (gRNA #1), UBE2G2 (gRNA #1) or UBE2J2 (gRNA #1). At 2 days after infection, gRNA-expressing cells were selected using puromycin. Cell surface expression of integrin-α4 (ITGA4), thrombomodulin (THBD) and HLA-I (HLA-A2 for THP-1 cells, HLA-A3 for U937 cells) was assessed by flow cytometry at 10 days (U937) or 15 days (THP-1) post infection. (C) THP-1 cells expressing US2 were lentivirally transduced with two CRISPR gRNAs (#4 and #5) targeting UBE2G2. At 2 days after infection, gRNA-expressing cells were selected using puromycin, and expression of integrin α1 (ITGA1), integrin α2 (ITGA2) or IL12 receptor β1-subunit (IL12R-B1) was assessed by flow cytometry at 7 dpi. (D) Lysates from cells used in B were prepared and subjected to immunoblotting analysis for total protein expression levels of ITGA2, ITGA4, thrombomodulin, and HLA-I (HCA2). US11-expressing cells were included as a control. Actin was used as a loading control. The asterisk marks an unspecific band.

Article Snippet: Antibodies Primary antibodies used in our studies were: mouse anti-HLA-I HC HC10 monoclonal antibody (mAb), 1:400; mouse anti-HLA-I HC HCA2 mAb, 1:25; phycoerythrin (PE)-conjugated mouse anti-HLA-A2 mAb (clone BB7.2, no. 558570, BD Pharmingen), 1:20; human anti-HLA-A3 OK2F3 mAb (LUMC, Leiden, the Netherlands), 1:40; mouse anti-TfR H68.4 mAb (no. 13-68xx, Invitrogen), 1:5000; mouse anti-FLAG-M2 mAb (no. F1804, Sigma-Aldrich), 1:10,000; rat anti-HA 3F10 mAb (no. 11867423001, Roche) 1:1000; rabbit anti-UBE2G2 mAb (EPR9248, no. ab174296, Abcam), 1:1000; rabbit anti-TRC8 polyclonal antibody (pAb) (H89, no. sc-68373, Santa Cruz Biotechnology), 1:1000; mouse anti-CD141 (THBD) mAb (clone 1A4, no. 559780, BD Pharmingen), 1:320; mouse anti-CD49b (ITGA2) mAb (clone 12F1, no. 555668, BD Pharmingen), 1:1000; mouse anti-CD49d (ITGA4) mAb (clone 9F10, no. 555502, BD Pharmingen), 1:1000; rabbit anti-ITGA4 mAb (EPR1355Y, no. ab81280, Abcam), 1:1000; mouse anti-IL12 receptor β1 subunit (CD212) mAb (clone 2.4E6, no. 556064, BD Pharmingen), 1:1000; and rabbit anti-ITGA2 mAb ( {"type":"entrez-protein","attrs":{"text":"EPR17338","term_id":"523383441","term_text":"EPR17338"}} EPR17338 , no. ab181548, Abcam), 1:1000.

Techniques: Expressing, Transduction, CRISPR, Plasmid Preparation, Infection, Flow Cytometry, Western Blot

Journal: Journal of Cell Science

Article Title: Multiple E2 ubiquitin-conjugating enzymes regulate human cytomegalovirus US2-mediated immunoreceptor downregulation

doi: 10.1242/jcs.206839

Figure Lengend Snippet: UBE2G2 and UBE2J2 regulate US2-induced immunoreceptor downregulation. (A,B) U937 (A) or THP-1 (B) cells expressing US2 were lentivirally transduced with a CRISPR/Cas9 vector targeting TRC8 (gRNA #1), UBE2G2 (gRNA #1) or UBE2J2 (gRNA #1). At 2 days after infection, gRNA-expressing cells were selected using puromycin. Cell surface expression of integrin-α4 (ITGA4), thrombomodulin (THBD) and HLA-I (HLA-A2 for THP-1 cells, HLA-A3 for U937 cells) was assessed by flow cytometry at 10 days (U937) or 15 days (THP-1) post infection. (C) THP-1 cells expressing US2 were lentivirally transduced with two CRISPR gRNAs (#4 and #5) targeting UBE2G2. At 2 days after infection, gRNA-expressing cells were selected using puromycin, and expression of integrin α1 (ITGA1), integrin α2 (ITGA2) or IL12 receptor β1-subunit (IL12R-B1) was assessed by flow cytometry at 7 dpi. (D) Lysates from cells used in B were prepared and subjected to immunoblotting analysis for total protein expression levels of ITGA2, ITGA4, thrombomodulin, and HLA-I (HCA2). US11-expressing cells were included as a control. Actin was used as a loading control. The asterisk marks an unspecific band.

Article Snippet: Primary antibodies used in our studies were: mouse anti-HLA-I HC HC10 monoclonal antibody (mAb), 1:400; mouse anti-HLA-I HC HCA2 mAb, 1:25; phycoerythrin (PE)-conjugated mouse anti-HLA-A2 mAb (clone BB7.2, no. 558570, BD Pharmingen), 1:20; human anti-HLA-A3 OK2F3 mAb (LUMC, Leiden, the Netherlands), 1:40; mouse anti-TfR H68.4 mAb (no. 13-68xx, Invitrogen), 1:5000; mouse anti-FLAG-M2 mAb (no. F1804, Sigma-Aldrich), 1:10,000; rat anti-HA 3F10 mAb (no. 11867423001, Roche) 1:1000; rabbit anti-UBE2G2 mAb (EPR9248, no. ab174296, Abcam), 1:1000; rabbit anti-TRC8 polyclonal antibody (pAb) (H89, no. sc-68373, Santa Cruz Biotechnology), 1:1000; mouse anti-CD141 (THBD) mAb (clone 1A4, no. 559780, BD Pharmingen), 1:320; mouse anti-CD49b (ITGA2) mAb (clone 12F1, no. 555668, BD Pharmingen), 1:1000; mouse anti-CD49d (ITGA4) mAb (clone 9F10, no. 555502, BD Pharmingen), 1:1000; rabbit anti-ITGA4 mAb (EPR1355Y, no. ab81280, Abcam), 1:1000; mouse anti-IL12 receptor β1 subunit (CD212) mAb (clone 2.4E6, no. 556064, BD Pharmingen), 1:1000; and rabbit anti-ITGA2 mAb ( {"type":"entrez-protein","attrs":{"text":"EPR17338","term_id":"523383441","term_text":"EPR17338"}} EPR17338 , no. ab181548, Abcam), 1:1000.

Techniques: Expressing, Transduction, CRISPR, Plasmid Preparation, Infection, Flow Cytometry, Western Blot

Journal:

Article Title: VLA-2 (?2?1) Integrin Promotes Rotavirus Entry into Cells but Is Not Necessary for Rotavirus Attachment

doi: 10.1128/JVI.76.3.1109-1123.2002

Figure Lengend Snippet: Flow cytometry histograms of the cell surface expression of integrin subunits α2 and β1 of VLA-2. The cell line analyzed is indicated in each histogram. Expression of α2 or β1 was determined by using 10 μg of MAb AK-7 (MA104, Caco-2, and CHO-p901) or MAb Ha1/29 (Rie1) or of MAb MAR4 (MA104, Caco-2, and CHO-p901) or MAb Ha2/5 (Rie1)/ml, respectively, in a single- or two-step stain. MAb NV3901, directed against the recombinant Norwalk virus capsid (35, 64), was used to match the isotype (IgG1) in a two-step stain.

Article Snippet: The purified MAb AK7 (IgG1) directed against the human α2 subunit (CD49b) of VLA-2, MAb MAR4 (IgG1) directed against the human β1 subunit (CD29) of VLA-2, MAb 9F10 (IgG1) directed against the human α4 subunit (CD49d) of VLA-4, MAb 23C6 (IgG1) directed against the human integrin complex αvβ3 (CD51/CD61), MAb VI-PL2 (IgG1) directed against the human β3 subunit (CD61) of αvβ3, MAb Hmα2 (hamster IgG; group 1; κ) directed against the mouse α2 subunit (CD49b) of VLA-2, MAb 9EG7 (rat IgG2a; κ) directed against the mouse β1 subunit (CD29) of VLA-2, MAb Ha1/29 (Armenian hamster IgG; group 2; λ) directed against the rat α2 subunit (CD49b) of VLA-2, and MAb Ha2/5 (Armenian hamster IgM; κ) directed against the rat β1 subunit (CD29) of VLA-2 were purchased from Pharmingen, San Diego, Calif. Additional antibodies for flow cytometry analysis, MAbs AK7, MAR4, HA1/29, and Ha2/5 conjugated to fluorescein isothiocyanate (FITC) or R-phycoerythrin (R-PE), goat polyclonal anti-mouse or anti-rat total Ig conjugated to FITC or R-PE, and a mouse anti-hamster (IgG groups 1, 2, and 3) MAb conjugated to R-PE, were also purchased from Pharmingen.

Techniques: Flow Cytometry, Expressing, Staining, Recombinant