Figure 2

S1P-specific inhibitor PF-429242 blocks CREB3L2 processing. (A) Diagram of CREB3L2 structure with proteolytic cleavage sites. Act domain, Activation Domain. bZIP, Basic Leucine Zipper Domain. TM, Transmembrane Domain. (B) In vitro generated human ASCs were treated with 10 μM PF-429242 or vehicle control (dH2O) on the day that appears first (d6, d8, d10) and protein lysates generated on the day that appears subsequently (d8, d10, d13) were analyzed by Western blotting. Intact and dashed arrows show migration of CREB3L2 full-length protein and cleaved C-terminus, respectively. (C) In vitro differentiated human B-cells were treated with 10 μM PF-429242 or vehicle control (dH2O) at day 6 and analyzed subsequently for phenotype on the indicated days. Live cells were evaluated for expression of B-cell markers CD19 and CD20 and ASC markers CD38 and CD138. Percentages of cells are shown within the quadrant gates. The experiment was performed with 4 donors, a representative donor is shown. (D) Samples treated as in part (B) were assessed for cell number using CountBright beads. (E) In vitro activated human B-cells from 4 donors were treated with indicated amount PF-429242 or vehicle control (dH2O) at day 6 and assessed for cell number at 48 h. Results are displayed as the percentage viable cells relative to control. Supernatants from samples in part (B) were analyzed by ELISA for production of (F) IgM and (G) IgG. Significance was determined by t-test.