Figure 4. DHX15 regulates Siah2 protein stabilities and its E3 ligase activity.

A) C4-2 cells were transfected with two different siRNAs targeting DHX15 or control for 72h. The mRNA expression of DHX15 and Siah2 were detected by qPCR. B) 293 cells were transfected with HA-Siah2, GFP and different dose of Flag-DHX15-WT or Flag-DHX15-K166A for 48h, cell lysates were subjected to western blotting and probed with anti-Flag and anti-HA antibodies. GFP was served as transfection efficiency control. C) 293 cells were transfected with HA-Siah2 or/and Flag-DHX15 as indicated for 48h and then treated with cycloheximide (CHX, 50ug/ml) for 1,3,6 h, and cell lysates were subjected to western blotting for detecting HA-Siah2 and Flag-DHX15 expression, GFP was served as control. The degradation curves of Siah2 by cycloheximide chase in the presence of DHX15 are shown on the right side. D) 293 cells were transfected with Flag-DHX15, Myc-AR and HA-Siah2 as indicated, the analysis was performed as described for (C) and the degradation curve of Myc-AR was presented. E) 293 cells were transfected with Flag-DHX15, Myc-Spry2 and HA-Siah2 as indicated, the analysis was performed as described for (D). F) C4-2 cells were transfected with siRNAs targeting DHX15 or/and Siah2 as indicated for 72 h and subjected to western blotting and probed with DHX15, Siah2 and Sprouty2 antibodies. G) PC3 cells were infected with lentivirus encoding for shRNA to DHX15 or Scramble, and cells lysates were subjected to western blotting and probed with DHX15, Siah2 and Spry2 antibody. Data represent average of a minimum of 3 technical replicates representative of 3 different experiments.