Figure 4.

PABPC1 regulates the production of secreted Ig in plasma cells. A, MPC11 cells were transduced with control shRNA (shCtrl) or two different shRNAs against Pabpc1 (shPabpc1.1 and shPabpc1.2, respectively), and expression of PABPC1 was analyzed by immunoblotting. B and C, knockdown PABPC1 decreased expression of the surface IgG2b in MPC11 cells. Expression of IgG2b on the cell surface or total IgG2b was analyzed through cell surface staining or intracellular staining in MPC11 cells, respectively. B, representative flow cytometry plots; C, summary of MFI of IgG2b staining from seven experiments. D–F, PABPC1 enhanced membrane Igγ2b while suppressing secreted Igγ2b expression in MPC11 cells. As in A, expression of secreted Igγ2b (D) or membrane Igγ2b (E) was analyzed by qRT-PCR, and ratios of the membrane Igγ2b to the secreted Igγ2b were calculated (F). G, PABPC1 promoted the membrane IgM expression in LPS-activated primary B cells. As in Fig. 3A, RNA was extracted from LPS-stimulated B cells transduced with retrovirus containing a scrambled sequence (shCtrl) or two independent shRNAs against Pabpc1 (shPabpc1) at 96 h after LPS stimulation. The expression of membrane Igμ was analyzed by qRT-PCR, and the ratios of membrane Igμ to secreted Igμ were calculated as indicated. H–J, shRNA-resistant PABPC1 reversed the defects of mIg/sIg ratio in the PABPC1-deficient MPC11 cells. H, Flag-tagged shRNA-resistant PABPC1 was transduced into MPC11 cells with Pabpc1 knockdown, and expression of exogenous (3xFLAG-PABPC1) and endogenous PABPC1 was determined by immunoblotting. I, expression of surface IgG2b was determined by flow cytometry analysis. J, the ratio of the membrane Igγ2b to the secreted Igγ2b mRNA was determined by qRT-PCR. The data are representative (A, B, H, and I) or summary (C–G and J) of at least three independent experiments. ***, p < 0.001; **, p < 0.01; *, p < 0.05 in Student's t test.