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. 2020 Mar 18;136(4):468–479. doi: 10.1182/blood.2019004147

Figure 4.

Figure 4.

Depletion of YWHAE inhibits translation initiation complex formation. (A-B) Western blot analysis in H929 (A) and KMS11 and JJN3 (B) MM cell lines infected with either scrambled (pLKO.1) or two 14-3-3ε-targeted shRNAs was performed using the indicated mAbs. (C) Western blot analysis in H929 (left) and KMS11 (right) 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. GAPDH ratio is shown (right). (D) m7GTP was used to pull down m7GTP binding proteins in H929 KO cells expressing pLenti6-empty (empty) or pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε). Pull-down products were subjected to western blot analysis and probed with the indicated antibodies. (E) Anti-FLAG antibody was used to pull down 14-3-3ε binding proteins in H929 KO cells expressing pLenti6-FLAG-YWHAE OE plasmid. Cell lysate from H929 with YWHAE KO cells expressing pLenti6-empty was used as negative control (empty). Pull-down products were subjected to western blot analysis and probed with the indicated antibodies. (F) Western blot analysis in H929 MM cells infected with either scrambled (pLKO.1) or two 14-3-3ε-targeted shRNAs (left), and in H929 KO cells expressing pLenti6-empty (empty) or pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε; right). One representative blot of 2 is shown.