Journal: NPJ Parkinson's Disease

Article Title: Novel co-culture model of T cells and midbrain organoids for investigating neurodegeneration in Parkinson’s disease

doi: 10.1038/s41531-025-00882-8

Figure Lengend Snippet: Antibodies used for flow cytometry analyses

Article Snippet: CD4 PE , mouse IgG2aκ , Miltenyi Biotec , VIT4 , 130-113-214.

Techniques: Flow Cytometry, Recombinant

Journal: PLOS Pathogens

Article Title: MARCH2, a T cell specific factor that restricts HIV-1 infection

doi: 10.1371/journal.ppat.1012330

Figure Lengend Snippet: MARCH2 fold expression change relative to mock normalized to GAPDH in (A) PMA-differentiated THP-1, H9, and 293T cells, (B) primary CD4+ T cells from three donors and (C) primary Monocyte Derived Macrophages (MDMs) from three donors treated with human IFN-β (500 units/ml) for 4 h, 8 h, 16 h, and 24 h. (D) MARCH2 fold expression relative to mock normalized to GAPDH in PMA-differentiated THP-1 cells infected with HIV-1 JR-CSF and H9 cells infected with HIV-1 NL4-3 at 4 h, 8 h, 16 h, and 24 h post infection (hpi). Mock indicates mock-treated (PBS). Graphs represent mean ± SD from n = 3 independent experiments.

Article Snippet: Cells were incubated with 1: 40 dilution of antibodies (PE mouse anti-human CD4 [RPA-T4], Invitrogen; APC mouse anti-human CD184/CXCR4 [12G5, Invitrogen; APC mouse anti-human CD195/CCR5 [NP-6G4, Invitrogen) or isotype controls (PE mouse IgG1 kappa [P3.6.2.8.1, Invitrogen]; APC mouse IgG2a kappa [eBM2a, Invitrogen]) for 30 minutes at 4°C, washed, fixed and acquired on BD LSRFortessa followed by data analysis using FlowJo version 10.8.0.

Techniques: Expressing, Derivative Assay, Infection

Journal: PLOS Pathogens

Article Title: MARCH2, a T cell specific factor that restricts HIV-1 infection

doi: 10.1371/journal.ppat.1012330

Figure Lengend Snippet: (A ) MARCH2 (M2) knockdown verification in H9 cells knocked down with a M2 specific (shM2) or a scramble control (shCtrl) shRNA. Fold expression change of human M2 and MARCH3 (M3) transcripts in M2 depleted H9 cells relative to shCtrl treated cells and normalized to GAPDH is shown. Bottom panels show western blots of endogenous M2 proteins in shCtrl- or shM2- treated H9 cells. GAPDH serves as loading control. (B) Endogenous M2 reduces HIV-1 replication in H9 cells. Wild type (shCtrl) or M2 depleted (shM2) H9 cells were infected with HIV-1 NL4-3 and harvested at the indicated time points. HIV-1 nef and env DNA levels were determined by RT-PCR normalized to GAPDH. ( C ) HIV-1 virions produced in H9 cells knocked down for endogenous M2 have higher Env levels. Western blots of virions produced in wild type (shCtrl) or M2 depleted (shM2) H9 cells probing with anti-gp120, anti-gp41 and anti-p24 antibodies. Densitometry analysis of gp120 and gp41 band intensity relative to shCtrl and normalized to p24 are shown below (n = 3). ( D ) Endogenous M2 is incorporated into nascent HIV-1 virions. Virus from C was used to detect endogenous M2 by western blots probing with anti-M2 and anti-p24 antibodies. (E) M2 and MARCH8 (M8) knockdown verification in THP-1 cells depleted for either M2, M8 or both M2 and M8. In the left panel, fold expression changes of human M2 and M3 transcripts in shM2 expressing THP-1 cells relative to shCtrl expressing cells and normalized to GAPDH. In the right panel, fold expression changes of human M2 and M8 transcripts in shM2, shM8 and shM2/M8 expressing THP-1 cells relative to shCtrl expressing cells normalized to GAPDH. Bottom panels show western blots of endogenous M2 proteins in shCtrl- or shM2, shM8, shM2/M8- expressing THP-1 cells. β-Actin serves as loading control. (F) Endogenous M2 has no effect on HIV-1 replication in THP-1 cells. PMA-differentiated THP-1 cells expressing shCtrl or shM2, shM8, both shM2 and shM8 were infected with HIV-1 AD8 and infected cells were harvested at the indicated time points. HIV-1 nef and env DNA levels were determined by qPCR and normalized to GAPDH. (G and H) Only HIV-1 virions produced in THP-1 cells depleted for endogenous M8 have elevated Env levels. PMA-differentiated THP-1 cells expressing shCtrl, shM2, shM8, or shM2/M8 were infected with HIV-1 AD8 , cell lysates and culture supernatants were harvested at 48 hpi. In G , culture supernatants were concentrated by ultracentrifugation and p24 normalized purified virions fractions were used for western blot analyses probing for gp120, gp41, p24. The graphs on the right show quantification analysis of virion associated gp120 or gp41 band intensities normalized to p24 band intensities relative to shCtrl conditions. In H , p24 normalized purified virions fractions were used to infect TZM-bl cells; luciferase levels were measured 48 hpi, and the percentage (%) of relative infectivity was determined with respect to virus produced in the presence of shCtrl conditions. ( I ) Primary CD4+ T cells were purified from 3 donors, activated, and transduced with lentiviruses expressing either shCtrl or shM2 followed by infection with HIV-1 NL4-3 . Infected cells were harvested at the indicated time points. HIV-1 nef and env DNA levels were determined by RT-PCR and normalized to GAPDH. ( J ) Primary CD14+ cells were purified from 2 donors, differentiated, and transduced with lentiviruses expressing either shCtrl or shM2, shM8 or both shM2/M8 followed by infection with HIV-1 AD8 . Infected cells were harvested at the indicated time points and analyzed as in I . In A , C, D, E and G , representative blots from 3 independent experiments are shown. Graphs in A , B , E , F , G , H represent mean ± SD from 3 independent experiments. Graphs in I and J represent mean ± SD from 3 and 2 human donors respectively. Statistical analysis in A , B , E , F , I and J performed using unpaired t-test (two-tailed). In H , statistical significance was determined using one-sample t-test (two-tailed) when comparisons were performed with shCtrl and unpaired t-test (two-tailed) between non-Ctrl conditions. ns, non-significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (days post infection, dpi).

Article Snippet: Cells were incubated with 1: 40 dilution of antibodies (PE mouse anti-human CD4 [RPA-T4], Invitrogen; APC mouse anti-human CD184/CXCR4 [12G5, Invitrogen; APC mouse anti-human CD195/CCR5 [NP-6G4, Invitrogen) or isotype controls (PE mouse IgG1 kappa [P3.6.2.8.1, Invitrogen]; APC mouse IgG2a kappa [eBM2a, Invitrogen]) for 30 minutes at 4°C, washed, fixed and acquired on BD LSRFortessa followed by data analysis using FlowJo version 10.8.0.

Techniques: Knockdown, Control, shRNA, Expressing, Western Blot, Infection, Reverse Transcription Polymerase Chain Reaction, Produced, Virus, Purification, Luciferase, Transduction, Two Tailed Test

Journal: eLife

Article Title: Paradoxical imbalance between activated lymphocyte protein synthesis capacity and rapid division rate

doi: 10.7554/eLife.89015

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human mouse CD4 Monoclonal Antibody (RPA-T4), PE-Cyanine7, eBioscience , Invitrogen , Cat#: 25-0049-42 RRID: AB_1659695 , FACS (1 μl per test).

Techniques: Transgenic Assay, Western Blot, Recombinant, Activation Assay, Virus, DC Protein Assay

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Human CD4, monoclonal antibody, PE-Cy7 conjugated (flow cytometry) , BD , BD Biosciences Cat# 557852, RRID: AB_396897.

Techniques: Recombinant, Flow Cytometry, Neutralization, Functional Assay, Western Blot, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software, Fluorescence, Marker