Ctla 4

Manufactured by R&D Systems

Sourced in United States

CTLA-4 is a cell surface receptor that is a member of the immunoglobulin superfamily. It is expressed on the surface of T cells and acts as an immune checkpoint, negatively regulating T cell activation and proliferation.

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Lab products found in correlation

Actin, by Cell Signaling Technology (2 mentions) Hif 1α no ab2185, by Abcam (2 mentions) Lps escherichia coli lps 0111 b4 no l4391, by Merck Group (2 mentions) Recombinant human hmgb1, by BioLegend (2 mentions) Anti mouse pd l1 neutralizing antibody, by BioLegend (2 mentions) Lactate, by Merck Group (2 mentions) Collagen 1, by Abcam (1 mentions) Enhanced chemiluminescence kit, by Cytiva (1 mentions) Polyvinylidene fluoride membrane, by Merck Group (1 mentions) Runx2, by Cell Signaling Technology (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Ripa lysis buffer, by Keygen Biotech (1 mentions) Lag 3, by R&D Systems (1 mentions) Cd160, by Thermo Fisher Scientific (1 mentions) Tigit, by Thermo Fisher Scientific (1 mentions) Tim 3, by BioLegend (1 mentions) Lsrii cytometer, by BD (1 mentions)

5 protocols using ctla 4

Specificity of Anti-PD-1 Antibodies

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Example 11

The specificity of anti-PD-1 antibodies was examined by detecting their binding to CD28 family members ICOS, CTLA-4 and CD28 (R&D System) using standard ELISA. Briefly, different concentrations of 67D9, c67D9, or hu67D9 were added to 96-well plates coated with ICOS, CTLA-4, CD28 or PD-1-Fc, followed by incubation at room temperature for 2 h. After washing, HRP-conjugated goat anti-human kappa (for detecting c67D9 or hu67D9) or HRP-conjugated goat anti-mouse kappa (for detecting 67D9) was added and the plates were further incubated for 1 h. Finally, TMB was added as a substrate for HRP, and the absorbance was measured at 450 nm. As shown in FIG. 5A-5D, each of the anti-PD-1 antibodies 67D9, c67D9, and hu67D9 bound to PD-I, but not to the other CD28 family members (ICOS, CTLA-4 and CD28).

US10981991B2. PD-1 antibodies and uses thereof (2021-04-20). ASIA BIOTECH PTE. LTD. [SG]. Inventors: Nana Gu [CN], Ke Shao [SG].

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Immunoblotting for Cell Signaling Proteins

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The antibodies to BMAL1 (no. 14020), CLOCK (no. 5157), TIM-3 (no. 83882), PKM2 (no. 4053), cleaved caspase-3 (no. 9661), STAT1 (no. 14994), phospho-STAT1 (Tyr701; no. 9167), and actin (no. 3700) were obtained from Cell Signaling Technology. The antibody to GPR81 (no. orb335576) was obtained from Biorbyt. The antibodies to HIF-1α (no. ab2185) and BMAL1 (no. ab3350) were obtained from Abcam. The antibody to PD-L1 (no. MAB90781), PD-L2 (no. AF1022), and CTLA-4 (no. AF476) were obtained from R&D Systems. LPS (Escherichia coli LPS 0111:B4; no. L4391) and lactate (no. L6661) were obtained from Sigma. Recombinant mouse IL-7 (no. 577808), recombinant mouse HMGB1 (no. 764006), recombinant human HMGB1 (no. 557804), and anti-mouse PD-L1 neutralizing antibody (no. 124329) were obtained from BioLegend.

Deng W., Zhu S., Zeng L., Liu J., Kang R., Yang M., Cao L., Wang H., Billiar T.R., Jiang J., Xie M, & Tang D. (2018). The Circadian Clock Controls Immune Checkpoint Pathway in Sepsis. Cell reports, 24(2), 366-378.

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Protein Expression Analysis by Western Blot

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Total protein was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer
(KeyGEN BioTECH, China) and separated by 8% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), then transferred onto polyvinylidene fluoride membranes
(Millipore, USA). After blocking nonspecific protein binding with 5% BSA, blots were
incubated with primary antibodies against CTLA4 (1:1000, R&D Systems, USA),
Collagen 1 (1:1000, Abcam, UK), Runx2 (1:1000, Cell Signaling Technology, USA) and
GAPDH (1:12000, Sanjian, China). After extensive washing with PBS containing 0.1%
Triton X-100, membranes were incubated with horseradish peroxidase (HRP)-conjugated
secondary antibody (1:5000, Zhongshan, China) for 30 min at room temperature. The
signals were visualized by ChemiDoc XRS (Bio-Rad, USA) using an Enhanced
Chemiluminescence kit (Amersham Biosciences, UK) and analyzed using the ImageJ2x
software (USA).

Dai F., Zhang F., Sun D., Zhang Z.H., Dong S.W, & Xu J.Z. (2015). CTLA4 enhances the osteogenic differentiation of allogeneic human mesenchymal stem cells in a model of immune activation. Brazilian Journal of Medical and Biological Research, 48(7), 629-636.

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Immunoblotting for Cell Signaling Proteins

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Multi-marker Profiling of T Cell Activation in PLHIV

Cited 1 time

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Expression of membrane bound IC and cellular activation markers in CD4+ and CD8+ T cells were measured by flow cytometry with cryopreserved peripheral blood mononuclear cells (PBMC) from participants on ART (SCOPE) (Table 1, Supplementary Table 1) as previously described (30 (link)). In brief, cryopreserved PBMC were thawed and stained with surface phenotypic markers (CD3 [Clone UCHT1], CD4 [clone S3.5, Invitrogen], CD8 [Clone RPA-T8], CD14 [Clone M5E2], CD19 [Clone SJ25C1], CD45RA [HI100], CD27 [Clone O323], CCR7 [Clone 3D12]) and immune checkpoints (PD-1 [Clone EH12.1], CTLA-4 [Clone BNI3], LAG-3 [Clone FAB2319F, R&D], TIGIT [Clone MBSA43, eBioscience], TIM-3 [Clone F38-2E2, BioLegend], CD160 [Clone By55, eBioscience], 2B4 [Clone C1.7]). All antibodies were purchased from BD Bioscience unless indicated otherwise. LSR II cytometer (BD Bioscience) was used for acquisition. FlowJo 9 was used for analysis.

Chiu C.Y., Schou M.D., McMahon J.H., Deeks S.G., Fromentin R., Chomont N., Wykes M.N., Rasmussen T.A, & Lewin S.R. (2023). Soluble immune checkpoints as correlates for HIV persistence and T cell function in people with HIV on antiretroviral therapy. Frontiers in Immunology, 14, 1123342.

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