Figure 7: HuR and KSRP form a complex and bind to the same element in the NPM 3′-UTR.

(a) IP coupled to RT-PCR (upper panel) or RT-qPCR (lower panel) experiments was performed to determine the association of KSRP and HuR with the NPM mRNA in C2C12 cells. For RT-qPCR, NPM mRNA levels are shown±s.e.m. of three independent experiments, ***P<0.0001 (t-test). (b) Supershift binding assay was performed to demonstrate that the radiolabelled NPM 3′-UTR as well as the P1 cRNA probe, unlike the P2 probe, can associate to a complex containing KSRP (indicated by an asterisk (*)). (c) Gel-shift binding assay was performed with 500 ng of purified GST or His-KSRP proteins and the indicated radiolabelled cRNA probes. *shown on gel indicates the location of shifted complexes. (d,e) IP experiments were performed on C2C12 cells expressing Rluc, Rluc-NPM-3′ and Rluc-NPM-3′-mut1 using the KSRP or IgG antibody. IP of KSRP (d) and association with reporter RNAs (e) were determined as described in Fig. 5b,c. (f) C2C12 extracts treated or not with RNase A were used for IP experiments with the anti-KSRP or -IgG antibodies. The binding of HuR to KSRP was then assessed by western blot. For unknown reasons, we observed a shift in the molecular weight of KSRP that we believe could be due to the IP of a post-translationally modified KSRP isoform. (g) Agarose gel demonstrating effectiveness of RNAse treatment. (h) In vitro GST pull-down assay demonstrating that HuR directly interacts with KSRP. Input lanes account for 10% of the reaction performed in the assay. (i,j) IP coupled to RT-qPCR experiments was performed using anti-KSRP (i) or HuR (3A2) (j) antibodies on total extract from C2C12 cells treated with siHuR (i) or siKSRP (j). NPM mRNA levels in the immunoprecipitates were standardized (as described in the Methods) and normalized to the corresponding IgG and input sample. The NPM mRNA levels in siHuR or siKSRP conditions were plotted relative to siCtr conditions±s.e.m. of three independent experiments, *P<0.01, **P<0.001 (t-test). All gels/blots shown in the figure are representative of three independent experiments (except for the gel shown in Fig. 7b, which is representative of two).