(C) IL-2 production by JK cells treated with 20 M U0126, 25 M PB3, 20 M TP, 5 M JMS-038 or JMS-053 and stimulated over night with PMA/Io. cytokine production than for early signaling at the Is usually. However, further research will be necessary to deeply understand the regulatory role of PRLs during lymphocyte activation and effector function. and 0.0001. We have previously shown the traffic of PRL-1 to the IS in CD71-made up of slow recycling endosomes, which polarize intracellular pools of the TCR to the Is usually [12,17]. Therefore, the traffic of the highly homologous PRL-3 was here investigated. We studied in JK cells the subcellular distribution of PRL-3 tagged with the green fluorescent protein (GFP-PRL-3). GFP-PRL-3 was expressed with the expected size and recognized by Xdh specific anti-PRL-3 immunoglobulins (Physique 1B). Consistently with data obtained for PRL-1, steady-state distribution of GFP-PRL-3 and CD71 revealed that GFP-PRL-3 trafficked to the recycling compartment (Physique 1C,D, control samples). We then addressed whether the recycling compartment had an active role in PRL-3 trafficking towards the plasma membrane. In order to study this issue, we took advantage of Brefeldin A (BFA), which Cabozantinib S-malate inhibits the conventional secretory pathway  and blocks the surface expression of CD71 in T cells . Consistent with the traffic of PRL-3 through the endosomal compartment, we observed higher co-localization between GFP-PRL-3 and CD71 after endosomal compartment compaction promoted by BFA treatment (Physique 1C,D). To evaluate the traffic of GFP-PRL-3 to the plasma membrane through the recycling compartment, we calculated in these samples the ratio of recycling compartment vs. plasma membrane protein. As expected, BFA clearly Cabozantinib S-malate hampered the expression of CD71 at the plasma membrane, as revealed by the increment of this ratio. By contrast, BFA treatment had a weak effect on the plasma membrane localization of GFP-PRL-3 (Physique 1E). In concordance with this, immunofluorescence experiments showed that distribution of the endogenous Cabozantinib S-malate PRL-3 to the membrane and the endosomal compartment was not affected by BFA treatment (Supplementary Physique S1). These data might indicate the presence of a transport of PRL-3 to the plasma membrane independent of the BFA-sensitive secretory pathway or a more stable half-life at the plasma membrane that should be investigated. Interestingly, the presence of PRL-3 in recycling endosomes suggests that PRL-3 molecules in transit through this endosomal compartment might be targeted to the Is usually during activation, as it has been previously shown for the TCR . 2.2. Delivery of GFP-PRL-3 to the IS The distribution of GFP-PRL-3 to the Is usually was studied in cognate interactions established by JK cells transfected with GFP-PRL-3 and SEE-loaded Raji APCs. To investigate the localization of GFP-PRL-3 in the polarized recycling compartment at the Is usually, JK and Raji cells were allowed to interact during 20 min in order to established mature interactions, which were then stained for CD71. Confocal microscopy showed a clear accumulation of GFP-PRL-3 at the Is usually (Physique 2ACC), where it co-localized with the polarized recycling compartment (Physique 2A,D). In concordance, time-lapse confocal microscopy showed the co-localization of GFP-PRL-3 and mCherry-CD3 at the endosomal compartment polarized to the Is usually (Supplementary Physique S2 and movie 1). Accumulation of GFP-PRL-3 was not observed in specimens made up of JK cells interacting with Raji cells non-loaded with SEE, indicating that the observed accumulation was specific to SEE cognate interactions (Supplementary Physique S3). The traffic of PRL-1 and PRL-3 to the endosomal compartment and the Is usually suggests that these enzymes might regulate the secretion of cytokines, in particular those Cabozantinib S-malate secreted to the Is usually, such as interleukin-2 (IL-2) . Nevertheless, PRLs might also regulate the delivery to the Is usually of intracellular pools of the TCR or signaling molecules Lck and LAT, which also travel to the IS in the endosomal compartment [17,22]. Open in a separate window Physique 2 Distribution of GFP-PRL-3 to the immunological synapse. (A,B) Representative cell conjugates of JK cells Cabozantinib S-malate interacting with SEE-loaded and CMAC (blue) labelled Raji cells. The green (pseudocolor) and red channels as well as the merged images are shown. The molecule stained and observed in the red channel is usually indicated. Calibration bar of.