SDS-PAGE was performed on protein extracts obtained from human CD4+ T cell activated in vitro as previously described. Western blotting was performed as previously described (Ricciardi et al., 2018 (link)). The following antibodies were used for Western blotting: rabbit polyclonal to phospho-rpS6 Ser235/236 (1:1000, Cell Signaling), rabbit polyclonal to phospho-rpS6 Ser240/244 (1:1000, Cell Signaling), mouse monoclonal anti-Vinculin (1:1000, Millipore), mouse monoclonal anti-Actin (1:1000, Sigma), mouse monoclonal anti-Puromycin (1:10000, Millipore). Chemiluminescent signals were detected using Amersham ECL Prime (GE Healthcare Life Sciences) and images were acquired using the iBright CL750 Imaging System (Thermo Fisher Scientific).
Where indicated, cells were treated with either 2 μM PP242 (Sigma-Aldrich) or 3 μM MNK inhibitor (Sigma-Aldrich) for 30 min after 48 hr of Dynabeads stimulation.
Where indicated, cells were treated with either 2 μM PP242 (Sigma-Aldrich) or 3 μM MNK inhibitor (Sigma-Aldrich) for 30 min after 48 hr of Dynabeads stimulation.
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