Western blots analyses

HeLa and Jurkat cells were lysed in TR3 buffer (3% SDS, 10% glycerol, 10 mM Na₂HPO₄) or RIPA buffer (1.6 mM Na₂HPO₄, 8.4 mM NaH₂PO₄, 0.1 M NaCl, 0.1% SDS, 0.1% Triton X-100), supplemented with 2.5 mg/ml sodium-deoxycholate (D6750-25G, Sigma-Aldrich), complete protease inhibitor cocktail (05056489001, Roche) and 1 mM sodium orthovanadate (A2196,0005, AppliChem) or phosphatase inhibitor cocktails (P2850, Sigma-Aldrich); and then centrifuged at 20000 × g for 15 min. Protein content was measured using the DC Protein Assay (5000116, Bio-Rad Laboratories) with bovine serum albumin (BSA) (23210, ThermoScientific) as a standard, and 20 or 60 μg protein was loaded for each lane.

To detect HuR oligomeric species by western blotting, HeLa cells were seeded in 6-well plates and transfected with plasmids carrying V5-tagged HuR WT or HuR Y5F (Supplementary Table S1), using Lipofectamine 3000 transfection reagent (L3000001, Invitrogen), as stated in the manufacturer's instructions. After 24 h, cells were lysed in 50 mM BisTris pH 7.2, 50 mM NaCl, 10% w/v Glycerol, 0.001% Ponceau S and 1% digitonin, supplemented with 2.5 mg/ml sodium-deoxycholate (D6750-25G, Sigma-Aldrich), complete protease inhibitor cocktail (05056489001, Roche) and 1 mM sodium orthovanadate (A2196,0005, AppliChem). For samples in reduced and denatured conditions, the buffer was supplemented with β-mercaptoethanol and preheated at 95°C for 5 min before gel loading. However, for samples in non-reduced and non-denatured conditions, β-mercaptoethanol was not added and the preheating step was omitted as described before (23). Then, samples were separated by SDS-PAGE (8%, 12% or 15% acrylamide) and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% BSA (A3294, Sigma-Aldrich) in Tris-Base buffered saline (TBS)-0.1% Tween-20 for 1 h at room temperature.

For HuR and HuR pY5 detection, primary rabbit anti-HuR pY5 (made by ProteoGenix against the peptide CMSNG(pY)EDHMAED; Supplementary Figure S1A, B) and mouse anti-HuR (WH0001994M2, Sigma-Aldrich) were used. For V5-tag detection, membranes were incubated overnight at 4°C with V5-tag primary antibody (1:1000) (R960-25, Invitrogen). The immunoreactive bands were detected using Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore). The chemiluminescent signal from immunoreactive proteins was detected in the ImageQuant LAS 4000 imaging system (GE Healthcare). Protein bands were quantified by densitometric analysis using ImageLab v6.1 (BioRad) or the open source image processing program ImageJ software and normalized to tubulin housekeeping protein expression (25).

To test the specificity of the antibody against HuR pY5, 385 μg/ml HuR proteins were incubated with 20 μg/ml kinase domain of JAK3 (provided by Prof. Patrick Celie at the Netherlands Cancer Institute), a kinase previously found to phosphorylate HuR at tyrosine residues (26), and 1.5 mM ATP for 20 h at 4 ºC in 20 mM HEPES, pH 7.4, supplemented with 1 mM MgCl₂ and 1 mM MnCl₂. Dephosphorylation was performed with 3 U/ml alkaline phosphatase (P7640, Sigma-Aldrich) incubated for 30 min at 37ºC.