Fig. 3: LAMP5 interacts with IRF4 and stabilizes the IRF4 protein.

A Analysis of immunoprecipitates of HA-tagged LAMP5 in IM-9 cells with mass spectrometry. The proteins identified are indicated. B Co-immunoprecipitation of LAMP5 with IRF4 in ARP-1 or IM-9 cells. C Co-immunoprecipitation of LAMP5 with IRF4 in HEK293T cells co-transfected with LAMP5 and IRF4 plasmids. D Pull-down of HA-LAMP5 with IRF4 in HEK293T cells. E Immunofluorescent staining of ARP-1 or IM-9 cells with DAPI and antibodies against LAMP5 and IRF4. Scale bar, 10 μm. F Immunofluorescent staining of myeloma patients bone marrow samples with DAPI and antibodies against CD138, LAMP5 and IRF4 (n = 5). Scale bar, 10 μm. G Schematic of the truncations including ΔDBD and ΔIAD fragments. H Western blotting showing different truncations of IRF4 (ΔDBD and ΔIAD) in HEK293T cells. I Pull-down of LAMP5 with different truncations of IRF4 (ΔDBD and ΔIAD) in HEK293T cells. J–M The expression of IRF4 or c-MYC in myeloma cells with reduced (shLAMP5) or increased (LAMP5) LAMP5 expression. Nontargeted shRNA (shCtrl) or control vector (Vec) served as control. N, O The expression of LDHA or KLF2 in myeloma cells with reduced (shLAMP5) LAMP5 expression. Nontargeted shRNA (shCtrl) served as control. P, Q Immunofluorescent staining of ARP-1 or IM-9 cells (shCtrl and shLAMP5) with DAPI and antibody against LAMP5 and IRF4. Scale bar, 10 μm. R Immunofluorescent staining of tumor tissues in Fig. 2F with DAPI and antibodies against LAMP5 and IRF4. Scale bar, 20 μm. S Correlation coefficient of the mRNA levels of LAMP5 and mRNA levels of IRF4 or c-MYC in myeloma patients (n = 559). The correlations were evaluated using Pearson coefficient. r, correlation coefficient. B–D, H, I, and M are representative of two independent experiments. E, J–L, and N–R are representative of three independent experiments. Data are averages ±SD. P value was determined by Pearson coefficient. J, K, L, N, O P values were determined using one-way ANOVA.