The antibodies used in this study were anti-pS38 (McBride et al., 2006 (link)), anti-AID (30F12 from Cell Signaling Technology or McBride et al. [2004] (link)), and anti–α-tubulin (Sigma-Aldrich). Anti-γH2AX (20E3), anti–histone H3 (D1H2), anti-CDC6 (C42F7), and phospho-substrate antibodies 9611, 2261, and 9621, all from Cell Signaling Technology. Anti-FLAG M2 agarose (Sigma-Aldrich) was used for Flag immunoprecipitation and eluted with Flag peptide. Immunoprecipitated AID or cell lysates (70 µg) were separated on 12% NuPAGE (Invitrogen), transferred to PVDF membrane, probed with anti-pS38 antibody, stripped, and probed with anti-AID and then anti–α-tubulin antibody. Band intensity was quantified with ImageJ, and the ratios of pS38 to AID or AID to H3 signals were calculated. Relative changes were normalized to control samples.
Protocol detail
Phosphorylation of Activation-Induced Cytidine Deaminase
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Agarose
Anti antibody
Antibodies
Cell
Flag peptide
Histone h3
Immunoprecipitation
Membrane
Pvdf
α tubulin
Corresponding Organization : The University of Texas MD Anderson Cancer Center
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Variable analysis
independent variables
- Anti-pS38 antibody
- Anti-AID antibody (30F12 from Cell Signaling Technology or McBride et al. [2004])
- Anti–α-tubulin antibody (Sigma-Aldrich)
- Anti-γH2AX (20E3) antibody
- Anti–histone H3 (D1H2) antibody
- Anti-CDC6 (C42F7) antibody
- Phospho-substrate antibodies 9611, 2261, and 9621 (all from Cell Signaling Technology)
- Anti-FLAG M2 agarose (Sigma-Aldrich) used for Flag immunoprecipitation and eluted with Flag peptide
- PS38 to AID ratio
- AID to H3 ratio
- Immunoprecipitated AID or cell lysates (70 µg) separated on 12% NuPAGE (Invitrogen) and transferred to PVDF membrane
- Positive controls: Not explicitly mentioned.
- Negative controls: Not explicitly mentioned.
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