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. 2019 Apr 2;9:225. doi: 10.3389/fonc.2019.00225

Figure 6.

Figure 6

BAG3 knockdown increases Ago2 phosphorylation at Ser387 and facilitates recruitment of Ago2 to IL6 mRNA. (A) Matrix assisted laser desorption ionization time-of-flight peptide mass spectrum showed that phosphorylation of Ago2 at Ser387 was increased in BAG3 knockdown BxPC3 cells. (B) Immunoprecipitation was performed using pan-Ser/Thr antibody, Ago2 phosphorylation was then analyzed using Western blot analysis. (C–E) BxPC3 cells were transfected with wild type (WT), mutation at Ser387 to alanine (S387A) or to aspartic acid (S387D) Ago2, Ago2 expression was confirmed by Western blot (C), IL6 mRNA was analyzed using real-time RT-PCR (D), enrichment of IL6 mRNA by Ago2 was performed using RIP with Flag antibody followed by real-time RT-PCR (E). (F) Biotinylated 3′UTR RNA segment of IL6 mRNA was used to pulldown lysates from BxPC3 cells transfected with WT, S387A or S387D Ago2 construct, and the pulldown materials were analyzed by western blotting analysis using the indicated antibodies. N.S., not significant; *P < 0.01.