Whole cell extracts were prepared from FACS-sorted WT and DKO RFP+ CD4+ T cells (Tregs) as described previously [20 (link)]. Sorted Tregs were incubated for 1 hr at 37°C with or without 50nM Bafilomycin A1 (B1793; Sigma) for blockade of lysosome function. Primary antibodies used for Western blot analyses were as follows: anti-LC3 A/B (D3U4C, Cell Signaling, 12741), anti-actin (I-19, SantaCruz, SC-1616), anti-SWAP-70 (Q-28, Santa Cruz Biotechnology, SC-81991), anti-LRG47/IRGM1 (A19, Santa Cruz Biotechnology, SC-11075), anti-GBP1-5 (H-300, Santa Cruz Biotechnology, SC-28579), and anti-Tubulin (D66, Sigma, T0198). Anti-DEF6 antibody was generated in rabbit as previously described [18 (link)].
Protocol detail
Whole Cell Extract Preparation from Tregs
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Actin
Anti antibody
Antibodies
Bafilomycin a1
Cd4 t cells
Cell extracts
Lysosome
Rabbit
Tubulin
Western blot analyses
Corresponding Organization :
Other organizations : Hospital for Special Surgery, Cornell University, New York City College of Technology, City University of New York, University of Alabama at Birmingham, Columbia University Irving Medical Center, New York Hospital Queens, NewYork–Presbyterian Hospital
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Variable analysis
independent variables
- Presence or absence of Bafilomycin A1 (50nM)
- Levels of LC3 A/B
- Levels of SWAP-70
- Levels of LRG47/IRGM1
- Levels of GBP1-5
- Levels of DEF6
- Levels of actin
- Levels of tubulin
- Positive control: Whole cell extracts from WT Tregs
- Negative control: Whole cell extracts from DKO Tregs
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