Figure 7.

PTBP2 knock-down impairs CSR and AID binding to activated switch regions in primary B cells. (a) Immunoblot analysis of cell extracts derived from activated splenic B cells transduced with shRNAs directed against PTBP2 or AID. (b) CSR to IgG1 was measured by flow cytometry. CSR in cells transduced with scramble shRNA was assigned a value of 100. Histogram represents mean of 7 independent experiments with error bars depicting standard deviation from the mean. p-values were determined by the Student's t-test. (c) Reduced Iγ-Cμ circle transcripts in PTBP2 knock-down cells. RNA derived from indicated cells was reverse-transcribed and 3-fold dilutions of cDNA analyzed for Iγ-Cμ circle transcripts by PCR. –RT represents PCR template in which reverse-transcriptase was omitted. (d-e) Impaired binding of AID to Sμ and Sγ1 in PTBP2 knock-down primary B cells. B cells were stimulated with anti-CD40 and IL-4 for 48 hours and then subjected to ChIP using AID or control-IgG antibodies. Levels of Sμ or Sγ1 in anti-AID ChIP samples were measured by quantitative real-time PCR. The graph depicts qPCR values expressed as relative ChIP units with scrambled assigned an arbitrary value of 100. ChIP units are derived from normalizing Ct values to input and then subtracting IgG ChIP Ct values as background. Results shown are the average of three independent experiments expressed with error bars representing standard deviation from the mean. p-values were determined by the Student's t-test.