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. 2017 Jun 13;292(29):12285–12295. doi: 10.1074/jbc.M117.794834

Figure 3.

Figure 3.

PABPC1 is not required for CD45 alternative splicing. A, Jurkat cells were transduced with PLKO.1 lentivirus containing either shPabpc1 or a Scrambled sequence, and expression of PABPC1 was determined by immunoblotting analysis. GAPDH was served as a loading control. B, PABPC1 was not required for activation-induced CD45 isoforms switching in Jurkat cells. As in A, Jurkat cells stably expressing the shRNAs against PABPC1 or Scramble were stimulated with PMA, and expression of CD45RA and CD45RO was determined by flow cytometry. C, naïve B cells were isolated from C57BL/6 mice and stimulated with 10 μg/ml LPS in the presence of IL-4 (10 ng/ml) and IL-5 (10 ng/ml). The activated B cells were then transduced with retrovirus containing three independent shRNAs against Pabpc1 (shPabpc1) or a scrambled sequence (shCtrl) at 16 and 40 h after stimulation, and 1 μg/ml puromycin was added into cultured cells at 48 h after stimulation. At 96 h after stimulation, the expression of PABPC1 was analyzed by qRT-PCR. D and E, PABPC1 was not required for CD45 splicing in activated B cells. As in C, RNA was extracted from unstimulated B cells, LPS-stimulated B cells (mock), LPS-stimulated B cells transduced with retrovirus containing a scrambled sequence (shCtrl), or three independent shRNAs against Pabpc1 (shPabpc1). CD45 splicing was analyzed by RT-PCR; PCR products corresponding to each splicing isoform of CD45 are indicated (D); and cell surface expression of CD45RA, CD45RB, CD45RC, and B220 (CD45RABC) was determined by flow cytometry (E). E, red indicates the cells transduced with shCtrl, and green, blue, and cyan indicate the cells transduced with one of the shPabpc1 shRNAs. F and G, hnRNPLL was required for exclusion of CD45RA exon. Jurkat cells were stably transduced with shRNA against either hnRNPLL (shLL) or a Scrambled sequence (shCtrl). Expression of hnRNPLL was determined by immunoblotting (F), and expression of CD45RA and CD45RO was determined by flow cytometry (G). H and I, the short but not the long isoform of hnRNPLL regulates CD45 splicing. A20 cells were transduced with control construct (mock) or long or short isoform of hnRNPLL (LL-long or LL-short), respectively, and expression of the hnRNPLL was analyzed by immunoblotting (H). CD45 splicing was analyzed by RT-PCR, and the PCR products corresponding to each splicing isoform of CD45 are indicated (I). The data are representative of at least three independent experiments.