Figure 3.

YBX1 interacts with, and is symmetrically dimethylated by PRMT5. (A) Representative co-immunoprecipitation (Co-IP) of Myc-YBX1 and Flag-PRMT5 shows that YBX1 and PRMT5 interact more strongly under IL-1β—stimulating conditions in HEK293 and (B) HT29 cells. Anti-Myc IP and Anti-Myc input images were obtained from the same blot with the same exposure. Anti-Flag IP images were obtained from the same blot that was stripped and reprobed with Anti-Flag antibody and developed at different exposures. First two lanes represent empty vector control stable lines compared to second two lanes which represent stable lines dually co-expressing Myc-YBX1 and Flag-PRMT5 constructs. Bottom panels, (A) and (B) show representative graphs of quantified western blot Myc co-IP images relative to input. (C) Western analysis to assess PRMT5 expression levels in cells expressing either Myc-YBX1 alone (Ctrl) or in combination with PRMT5 overexpression vector or shRNA-PRMT5 constructs. (D) Western analysis to assess dimethylation status of immunoprecipitated Myc-WT-YBX1 in cells with PRMT5 overexpression or its shRNA knockdown (shPRMT5), respectively. Anti-SDMA IP and Anti-Myc IP images were obtained from the same blot, stripped and reprobed for total Myc but developed at different exposures. Input images were obtained from different blots and developed at different exposures. (E) Immunofluorescence showing the localization of Flag-WT-YBX1 and PRMT5 with and without IL-1β treatment (10 ng/ul). Hoechst was used for nuclear staining. Images were taken at 63 × . *p < 0.05 Ratio of Myc-YBX1/Flag-PRMT5 Co-IP vs.YBX1/Flag-PRMT5 Co-IP + IL-1β stimulation relative to input. Densitometry analysis was performed using ImageJ software.