Journal: Journal of immunology (Baltimore, Md. : 1950)
Article Title: Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Igk Feedback Inhibit V κ Recombination to Enforce Igκ Allelic Exclusion.
doi: 10.4049/jimmunol.2100696
Figure Lengend Snippet: FIGURE 2. Nemo inactivation leads to increased RAG cleavage of Igk loci. (A) Schematic for Southern analysis of Igk cleavage by RAG indicating the GL configuration of the Jj region of the Igk locus with flanking EcoRI and SacI restriction sites, the GL Jj region generated by EcoR1 and SacI digestion, and each Jj CE generated by RAG cleavage. (B) Representative Southern blot analysis of Jj cleavage and (C) quantification of the remaining GL Jj fragment in primary preB cell cultures from BCL2:IgH:Artemis−/−:Nemoflox/flox (BIA) and Mb1Cre1:BCL2:IgH:Artemis−/−:Nemoflox/flox (BIA:Nemo−/−) mice. Cells were collected after 4 d of culture in IL-7 or at 24, 48, or 72 h after IL-7 withdrawal of cultures treated with or without the ATM inhibitor (ATMi) KU55933 (15 lM). The probe 39 of the Jj region depicts the GL Jj fragment and indicated Jj CEs. Southern blot using a probe for Hprt was used to normalize DNA content in each lane. The quantification is the amount of GL Jj relative to Hprt and normalized to the BIA IL-71 sample. These data are from one indepen- dent experiment. (D) Schematic of TaqMan quantification of GL Jj indicating the relative positions of the primers and TaqMan probe used for each Jj gene segment. (E) TaqMan quantification of GL Jj1 from the same samples as the ones used for the Southern blot analysis in (B) and (C). TaqMan quantification of the Cd19 locus was used to normalize DNA content. The Jj1 value was first normalized to Cd19 and then to the BIA IL-71 sample. These data are from one independent experiment. (F) TaqMan quantification of GL Jj1 from an aliquot of the same samples as Fig. 1. These samples are from primary preB cell cultures from BCL2:IgH:Rag1−/−:Nemoflox/flox (BIR1), Mb1Cre1:BCL2:IgH:Rag1−/−:Nemoflox/flox (BIR1:Nemo−/−), BCL2:IgH:Artemis−/−:Nemoflox/flox
Article Snippet: These bone marrow cells were cultured for 4 d in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, 13 nonessential amino acids, 1 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin streptomycin, 50 mM 2-ME, and 5 ng/ml IL7 (407-ML; R&D Systems).
Techniques: Generated, Southern Blot
PeproTech is a verified supplier 
