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. 2014 Oct 16;42(20):12483–12497. doi: 10.1093/nar/gku953

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Cytoplasmic hnRNPA1 associates with 14-3-3 subunits and is sumoylated upon stimulation with FGF-2. (A, C, G and H) HEK293 cells were transiently transfected with (A) wild-type (WT) or S4AS6A mutant hnRNPA1-FLAG or (C, G and H) siRNAs against the indicated proteins or a non-targeting control (NT) were treated ± FGF-2 for the indicated time (A and G) or 60 min (C) and the cytoplasmic fraction analysed by SDS-PAGE/western blotting (WB). (A, G and H lower panels and C) Band intensities for hnRNPA1 were quantified and normalized to that for tubulin. Results show average of triplicate experiments ± SEM normalised to the corresponding controls. In Fig6A, lower panel shows quantification for both endogenous and the FLAG-tagged hnRNPA1. (B) HEK293 cells were treated with FGF-2 and the endogenous 14-3-3 protein subunits σ, θ or ϵ immunoprecipitated (IP) from the cytoplasmic fraction. Immunoprecipitates were analysed by SDS-PAGE/WB for endogenous hnRNPA1 or 14-3-3 proteins. (D) Endogenous hnRNPA1 was immunoprecipitated from the cytoplasmic fraction of HEK293 cells treated ± FGF-2. Immunoprecipitates were analysed by SDS-PAGE/WB with either Sumo1 or hnRNPA1 antibodies. The arrows indicate sumoylated-hnRNPA1. (E) HEK293 cells stably expressing HA-Sumo1 were treated with siRNA for 14-3-3σ, θ or NT before stimulation with FGF-2. HA-SUMO1 was immunoprecipitated with anti-HA antibodies from the cytoplasmic fraction and the samples analysed by SDS-PAGE/WB for the indicated proteins. (F and I) HEK293 cells stably expressing HA-SUMO1 were transiently transfected with FLAG-tagged WT or S4AS6A or K183R mutant hnRNPA1 and treated with FGF-2. Cytoplasmic fractions were immunoprecipitated for HA-SUMO1 and analysed as above. All experiments are representative of three independent experiments. See also Supplementary Figure S3.