FIGURE 5 from The Genetic Landscape of Ocular Adnexa MALT Lymphoma Reveals Frequent Aberrations in NFAT and MEF2B Signaling Pathways

Effects of CABIN1 deletion and mutations on the NFAT and MEF2B transcriptional activities. A, Western blot analysis of CABIN1 expression in WT SSK41 cells or in SSK41 cells transduced with lentiviral vectors expressing either a control shRNA or CABIN1-specific shRNAs. The expression of the housekeeping gene GAPDH was used as a loading control. B, Luciferase reporter assay for NFAT (top) and MEF2 (bottom) transcriptional activity in SSK41 cells transduced with lentiviral vectors expressing either a control shRNA or CABIN1-specific shRNAs. Where indicated, cells were stimulated for 4 hours with α-IgM F(ab’)₂. *, P = 0.001; **, P < 0.0002. C, Western blot analysis of CABIN1 expression in WT SSK41 cells or in SSK41 cells in which CABIN1 was initially knocked down using 3′-UTR targeting shRNA followed by expression of HA-tagged CABIN1 WT or mutants. CABIN1 was detected using anti-CABIN1 and anti-HA antibodies, while expression of the housekeeping gene GAPDH was used as a loading control. D, Luciferase reporter assay for NFAT (top) and MEF2 (bottom) transcriptional activity in SSK41 CABIN1 KD cells and cells expressing indicated CABIN1 constructs as shown in C. Where indicated, cells were stimulated for 4 hours with α-IgM F(ab’)₂, in comparison to stimulated HA-WT: *, P < 0.005; **, P < 0.0001; ***, P = 0.00006. E, Representative immunoprecipitation (IP) assays with anti-HA antibodies using whole-cell protein extracts from unstimulated and α-IgM F(ab’)₂–stimulated SSK141 cells expressing different CABIN1 mutants, as shown in C followed by Western blotting using indicated antibodies. Also shown mean and SD of relative SIN3A densitometry adjusted to immunoprecipitated CABIN1 in each cell type versus WT cells from three independent experiments. Statistical analyses of relative densitometry in mutants versus WT cells. *, P < 0.05; **, P < 0.01.