Fig. 1

HuR interacts with TERC in vitro. a RNA pull-down assays were performed using HeLa cell lysates and in vitro-transcribed RNAs depicted in Supplementary Fig. 1a. The presence of HuR in the pull-down materials was assessed by western blot analysis. p16 3′-UTR and CR (coding region) served as positive and negative controls, respectively. A 5-µg aliquot input (Inp.) and binding to GAPDH RNA were also assessed. b Purified his-HuR and in vitro-transcribed TERC was used for UV-crosslinking rEMSA assays. The covalently bound HuR was detected by western blotting. c Left, the association of HuR with TERC variants bearing mutations U40A, U100A, or U40A + U100A (Supplementary Fig. 1b) was determined by using RNA pull-down assays, as described in Fig. 1a. Right, quantification of the bands on the western blot (left); data are the means ± SD of the signals from three independent experiments and significance was analyzed by Student’s t-test (**p < 0.01). d In vitro ITC measurements of direct interactions between HuR and RNA oligonucleotides derived from human TERC, AUUUUUUGUCU (positions 37–47) and GUUUUUCUCG (positions 98–107), as described in Methods section. The dissociation constant (K_d) is calculated from 12 titrations and is indicated in the graphs