mAbs specific for mouse CD3 (clone 145-2C11), CD28 (clone 37.51), or CTLA-4 (clone UC10-4B9) were purchased from Biolegend, as were flourophore-conjugated anti-CD4 (Clone GK1.5), -CD8 (53-6.7), -Foxp3 (clone FJK-16s), -CD25 (Clone PC61), -CD44 (Clone IM7) and –GITR (Clone DTA-1) mAbs. Anti-human CD3 mAb (OKT3) was purified in-house. Polyclonal anti-PKC-θ (sc-212), anti-PKC-η (C-15 and sc-215), anti-PAK (N-20 and sc-882), anti-NFATc1 (7A6), anti-lamin B (M-20), and anti-α-tubulin (TU-02) Abs were obtained from Santa Cruz Biotechnology. Anti-p65 (NF-κB), anti-GIT2 and -αPIX Abs were obtained from Cell Signaling Technology. Anti-Foxp3 Abs (clone 150D/E4 for immunoblotting and clone FJK-16s for flow cytometry) were purchased from eBiosciences. Alexa-647-conjugated anti-mouse Ig and Alexa-555-conjugated anti-rabbit Ig were obtained from Molecular Probes. Digitonin was obtained from EMD Chemicals. Calf intestinal alkaline phosphatase was purchased from New England Biolabs. Recombinant CD86-Fc was previously described46 (link).
Protocol detail
Antibody Characterization for Murine T-cell Studies
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CTLA-4 (clone UC10-4B9)
–GITR (Clone DTA-1)
anti-lamin B (M-20),
anti-α-tubulin (TU-02)
Anti-Foxp3 Abs (clone 150D/E4
clone FJK-16s
Alexa-647-conjugated anti-mouse Ig
Alexa-555-conjugated anti-rabbit Ig
Calf intestinal alkaline phosphatase
Alkaline phosphatase
Cd25
Cd44
Clone
Ctla 4
Digitonin
Flow cytometry
Git2
Gitr
Human
Intestinal
Lamin b
Molecular probes
Mouse
Nf κb
Okt3
Pkc θ
Rabbit
α tubulin
Corresponding Organization :
Other organizations : La Jolla Institute For Allergy & Immunology, Scripps Research Institute, Japan Science and Technology Agency, RIKEN Center for Integrative Medical Sciences, National University of Singapore
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Variable analysis
independent variables
- MAbs specific for mouse CD3 (clone 145-2C11)
- MAbs specific for mouse CD28 (clone 37.51)
- MAbs specific for mouse CTLA-4 (clone UC10-4B9)
- Foxp3 expression
- CD4, CD8, CD25, CD44, GITR expression
- Activation of signaling proteins (PKC-θ, PKC-η, PAK, NFATc1, NF-κB, GIT2, αPIX)
- Anti-human CD3 mAb (OKT3) used as control
- Lamin B and α-tubulin used as loading controls for immunoblotting
- Digitonin, calf intestinal alkaline phosphatase, and recombinant CD86-Fc used as reagents
- Anti-human CD3 mAb (OKT3)
- No negative controls explicitly mentioned
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