Figure 3.

14-3-3ε interacts with and affects the mTORC1 signaling pathway. (A-B) Anti-FLAG antibody was used to pull down 14-3-3ε binding proteins in H929 KO cells expressing pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε). Cell lysate from H929 (FLAG-negative with YWHAE WT) cells was used as negative control. Pull-down products were subjected to mass spectrometry or western blot analysis. (A) Hallmark GSEA of 14-3-3ε interacting proteins detected by mass spectrometry analysis. (B) Pull-down products were subjected to western blot analysis and probed with the indicated antibodies. (C) Western blot analysis in H929 MM cells infected with either scrambled (pLKO.1) or two 14-3-3ε-targeted shRNAs was performed using the indicated mAbs, including 14-3-3ε mAb to confirm KD efficiency. (D) Western blot analysis in H929 KO cells expressing pLenti6-empty (empty) or pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε) was performed using the indicated mAbs, including 14-3-3ε to confirm addback efficiency. GAPDH was used as loading control. One representative blot of 2 is shown. (E) Pearson correlation coefficients between YWHAE and individual gene expression levels across all patients with MM were calculated; the resulting rank-ordered gene list was subjected to GSEA. GSEA false discovery rate (FDR)-q values and normalized enrichment scores for genes significantly correlated with YWHAE expression in primary MM cell RNA-seq data are shown in the graph. (F) GSEA-derived enrichment plots for mTORC1 and UPR pathways.