Figure 2.

PTBP2 knock-down impairs CSR. (a) shRNAs efficiently knock-down PTBP2 expression in CH12 cells. CH12 cells were lentivirally infected with shRNAs against PTBP2 (PTBP2-1, PTBP2-2) or non-specific control (scramble). Whole cell extracts derived from unstimulated (U) or CIT- stimulated (S) cells were analyzed on immunoblots using anti-PTBP2 or control GAPDH antibodies. (b) PTBP2 knock-down or control CH12 cells were stimulated with CIT for 72 h and switching to IgA was measured by flow cytometry. A representative histogram is shown and the percentage of IgA-positive cells is indicated. (c) Quantification of CSR in PTBP2 knockdown cells. CSR in control cells was assigned an arbitrary value of 100 for each experiment. The data shows the mean of 5 independent experiments with error bars representing standard deviation from the mean. P-values were determined by the Student's t-test. (d) Reduced amounts of circle transcripts in PTBP2 knock-down cells. 3-fold dilutions of cDNA generated by reverse-transcription were amplified by PCR for I_α-C_μ or β-actin (control) transcripts. –RT represents PCR from template in which reverse-transcriptase was not added. (e) Enforced expression of PTBP2 in knock-down cells. CH12 cells expressing scrambled or PTBP2-2 shRNA were transduced with empty vector or vector harboring PTBP2 cDNA and cell extracts were analyzed by immunoblotting. (f) PTBP2 expression rescues CSR. PTBP2 knock-down cells expressing PTBP2 through a lentiviral construct were stimulated with CIT and CSR to IgA was measured by flow cytometry. CSR frequency in scramble was assigned a value of 100 in each experiment. The data represents the mean of 3 independent experiments with error bars representing standard deviation from the mean.