Alexa594 wga

Immunostainings were performed using standard protocols. The following primary antibodies were used: rabbit anti-SKTL at 1:20000 (Claret et al., 2014 (link)); mouse anti-Myc 9E10 (Santa Cruz) at 1:250; rabbit anti-RAB11 at 1:8000 (Nakamura); rabbit anti-RAB5 at 1:50 (Marcos Gonzalez-Gaitan); guinea pig anti-HRS at 1:500 (H. Bellen); mouse anti-KDEL at 1:300 (abcam 10C3, RRID: AB_298945); rabbit anti-NUF at 1:500; mouse anti-spNF at 1:50 (Abdu Uri); rabbit anti-Syntaxin16 at 1:1000 (R.Leborgne); rabbit anti-PKC (Santa Cruz) at 1:1000; mouse anti-HTS (Hybridoma, RRID: AB_528289) at 1:10; mouse anti-ß-Gal (Promega) at 1:250; rabbit anti-Staufen at 1:200. F-actin was visualised after staining with rhodamine-phalloidin (RRID: AB_2572408) or Alexa488-phalloidin (RRID: AB_2315147; life technology) at 1:200. Alexa594-WGA (Molecular probes) was used at 1:100 to stain nuclear membrane. Images were obtained with a ZEISS LSM700 confocal microscope and a Leica TCS-SP5 AOBS inverted scanning microscope.

Antibodies used were: monoclonal and polyclonal anti-FLAG antibodies from Sigma-Aldrich, polyclonal anti-Alix from Covalab, monoclonal anti-actin from Millipore, polyclonal antibodies against EGFR, Myc and EEA1 were from Santa Cruz, anti-GFP and anti-LAMP1 from Abcam, monoclonal anti-Rab11, monoclonal anti-clathrin and anti-GM130 from BD transduction, monoclonal anti-β1 (9EG7) was from BD Pharmingen, anti-STAT5 and anti- phosphorylated STAT5 were from Cell Signaling. Monoclonal anti-LBPA (6C4) and polyclonal anti-endophilin A antibodies were generous gifts from Jean Gruenberg (Geneva, Switzerland) and Pietro De Camilli (New Haven, United States) respectively. Monoclonal anti-IL2Rβ antibody (mAb 341) was described in39 (link). Secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 594 and Alexa 594-WGA, as well as EGF-TRITC, CTxB-TRITC, Tf-TRITC and Dextran-TRITC were from Molecular Probes. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies were from Jackson Laboratories. Filipin, chlorpromazine, EGF, bafilomycin A1 were from Sigma-Aldrich. 6 nm BSA-Gold tracer was from Aurion (The Netherlands).