Fig. 5

RNA-IP for SLC4A4 ARE-forming mutation. HEK 293 cells were co-transfected with HA-ZFP36 expression vector along with either WT SLC4A4 3'UTR or Mut SLC4A4 3'UTR. Following transfection, cells were lysed and incubated with either anti-HA-ZFP36 or anti-Myc antibodies. The pulled down RBP-mRNA immune-complex with either anti-HA-ZFP or the control anti-Myc complexes were processed for immunoblotting (upper panel) or for cDNA RT-QPCR (lower panell). First lane in the blot corresponding to total lysate was not subjected to immunoprecipitation. The mRNA levels were normalized to RPLPO as background control. Specific enrichment was calculated by reporter mRNA levels/background (RPLPO) mRNA levels and further normalized to negative control (anti-Myc) as 1.0. Data are from one representative experiment with two RT-QPCR reactions (each in triplicate). Data are mean ± SEM. Statistical significance was assessed by two-way ANOVA and Student’s t-test; **P < 0.001, ***P < 0.0001 as indicated