Figure 4: HuR binds more MYC mRNA in miR cells.

(a) HuR-immunoprecipitations (HuR-IPs) coupled with RT–qPCR analysis revealed higher level of MYC mRNA precipitated by the HuR in miR cells compared with control. Histogram represents mean±s.e.m. (the control is set as 1; n=3; t-test, unequal variances, one-tail, **P≤0.01). (b) Western blot analysis of HuR, Ago2, MYC and Vcl (negative control) upon RNA pull-down assay. Biotinylated RNAs corresponding to the fragments B and D of MYC 3′ UTR (see Supplementary Fig. 6a) were used for pull-down assays with extracts from control and miR cells. Different exposure times were used for input and pull-down results. (c) Cytoplasmic extracts from control and miR-17-19b-overexpressing cells were subjected to IPs using anti-HuR antibody (ab). Each HuR-IP was loaded as two samples: 50% IP was probed with anti-HuR and 50% with pS/T ab. The anti-HuR ab precipitated both, the full length (35 kDa) and cleaved form 1 (CP-1, 25 kDa), of which the last is detected as predominantly phosphorylated. A mild, yet reproducible decrease of phospho-HuR was measured in miR cells when compared with control (histogram, right panel). Quantification represents mean of pS/T signal normalized to CP-1±s.e.m. (control cells are set as 1; n=3; t-test, one-tail, unequal variances, *P≤0.05). See related Supplementary Fig. 6.