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. 2014 Jun;34(12):2135–2146. doi: 10.1128/MCB.00281-14

FIG 4.

FIG 4

Apo-CRABP2 associates with HuR and enhances its affinity for target mRNAs. For all experiments, cells were cultured in delipidated medium for 48 h to deplete retinoid stores. (A) Lysates from MCF-7 cells were treated with vehicle or RNase (100 μg/ml), CRABP2 was immunoprecipitated, and precipitates were immunoblotted for the presence of CRABP2 and HuR. (B) Lysates from MCF-7 cells were treated with vehicle or RNase, HuR was immunoprecipitated, and precipitates were immunoblotted for the presence of HuR and CRABP2. (C) M2−/− cells were transfected with a vector encoding Flag-CRABP2 and treated or not with CGP74514A (2 μM for 2 h). Flag-CRABP2 and HuR were detected by immunostaining, and cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Bars, 5 μm. (D) MCF-7 cells and M2−/− cells expressing Flag-tagged CRABP2 were treated with CGP-74514A (2 μM for 2 h). Endogenous CRABP2 in MCF-7 cells and Flag-tagged CRABP2 in M2−/− cells were visualized by immunostaining using CRABP2 and Flag antibodies, respectively. Cells were also immunostained for HuR and counterstained with DAPI. Confocal fluorescence microscopy was used to visualize cells. Bars, 5 μm. (E) HuR was immunoprecipitated from M2−/− cells stably overexpressing EGFP or EGFP-CRABP2 (Fig. 1A). Apaf1, Elavl1, and Actb mRNAs coprecipitating with HuR were detected by semiquantitative PCR. (F) Coomassie blue-stained gels visualizing recombinant, bacterially expressed, and purified His-CRABP2 and His-CRABP2ΔNLS. (G) Fluorescein-labeled RNA containing 39 bases corresponding to site 3 in the Apaf-1 3′ UTR was titrated with recombinant GST-tagged HuR alone or in complex with CRABP2 or CRABP2ΔNLS. To ensure saturation, CRABP2s were precomplexed with HuR at a 4:1 molar ratio. Progress of titrations was followed by monitoring the increase in the fluorescence anisotropy of the labeled RNA (λexcitation = 494 nm; λemission = 518 nm). Data representative of 3 independent experiments are shown. (Inset) Fluorescein-labeled RNA was titrated with CRABP2 or CRABP2ΔNLS.