Figure 8: The HuR-RRM3 motif is required for the formation of KSRP/HuR complex.

(a) Schematic diagram depicting the primary structure of HuR protein shows the three RNA-binding domains (RRM1-3) and the HNS. Also shown are the different HuR isoforms conjugated to the GFP tag. (b–e) Exponentially growing C2C12 muscle cells were transfected with GFP, GFP-HuR, GFP-RRM1-RRM2, GFP-HNS-RRM3, GFP-HNS or GFP-RRM3 plasmids. Total cell extracts (b,d) or total RNA (c,e) were prepared from these cells 24 h post transfection. (b) IP experiments on these cell extracts were performed using the KSRP antibody or IgG as a control. The input (10% of the total extract) and the immunoprecipitate were analysed by western blot using antibodies against GFP. (c) RT-qPCR analysis of total RNA prepared from these cells was performed using specific primers for NPM, MyoD and GAPDH mRNAs. NPM and MyoD mRNA levels were standardized against GAPDH mRNA and plotted relative to the GFP control condition±s.e.m. of three independent experiments. (d) IP experiments were performed using the GFP antibody on extracts from the C2C12 cells expressing GFP, GFP-HuR, GFP-RRM1-RRM2 or GFP-RRM3 and analysed by western blot using the GFP antibody. (e) RNA was isolated from the IP as described in d and RT-qPCR was performed using primers specific for NPM and RPL32 mRNAs. NPM mRNA levels were standardized against RPL32 mRNA levels. For each IP sample, NPM mRNA levels were normalized as described in Fig. 5 and plotted relative to the GFP IP±s.e.m. of three independent experiments.