IGF1R Endocytosis in Ewing Sarcoma Figure 7. Simplified model for IGF1R signaling in ES. Globally, after ligand binding IGF1R can signal directly from the membrane (IGF1R signaling internalization-independent), with a major degree of IRS1/PI3K/AKT signaling activation and a smaller enhancement of the Ras/Raf/MEK/ERK signaling pathway, or, when internalized either by clathrin and/or caveolin-dependent mechanisms, it can signal from vesicles. In the case of clathrin- dependent endocytosis, IGF1R is recruited to CCPs through interaction of its cytoplasmic tails with the protein adaptor AP2, which in turn recruits clathrin to the plasma membrane and promotes its assembly into a lattice network. Through protein-protein interaction, clathrin and AP2 also bind several proteins of the endocytic machinery, such as dynamin, which regulates the fission of the CCPs with the consequent formation of CCVs, and GRB2, which recruits and binds the SOS protein and the IGF1R-phosphorylated protein Shc, activating the RAS/RAF/MEK/ERK and p38MAPK signaling pathways. To a lesser extent, it also activates the IRS1/PI3K/AKT signaling pathway. In the case of caveolin-dependent endocytosis, IGF1R is recruited to caveolae, promoting caveolin phosphorylation and the subsequent recruitment of dynamin, which promotes caveolar budding and the formation of cavicles. Cav-1 phosphorylation phosphorylates IRS-1, enhancing the activation of thePI3K/AKT signaling pathway. The thickness of the arrows indicates the prevalence of signaling pathway activation. CPMZ treatment inhibits the formation of CCP by inhibiting clathrin-heavy-chain conformations and MCB inhibits the formation of cavicles by depleting cholesterol, disrupting the caveolae. doi:10.1371/journal.pone.0019846.g007 fixed with 2%(w/v) formaldehyde (Sigma-Aldrich) for 30 min. Cells were permeabilized in PBS/Triton x-100 0,1% (Invitrogen) for 30 minutes, incubated with PBS 50 nM NH4CL for 10 minutes, blocked for 30 minutes with PBS 0.2% BSA (Sigma Aldrich), and incubated overnight at 4uC with one of the following primary antibodies; rabbit polyclonal antibody against caveolin1 (Abcam), or rabbit polyclonal antibody against clathrin (Abcam), or mouse monoclonal antibody against clathrin (Affinity Bioreagents). After three washes in PBS, the slides were incubated with one of the following secondary antibodies: chicken anti- rabbit AlexaFluor594 (Invitrogen) or goat anti-rabbit Cy3 (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) or goat anti-mouse AlexaFluor594 (Invitrogen), for 45 min, and washed three times. Then, all the slides were incubated with rabbit polyclonal antibody against IGF1R (cell signalling) for 1 hour, washed three times, and incubated with chicken anti- rabbit Alexa 488 (Invitrogen) for 1 hour. Following this, cells were washed three times and nuclear DNA was counterstained with DAPI (2.5 mg/mL) (Invitrogen). Cells were mounted using the Slowfade Gold Antifade reagent (Invitrogen) or Vectashield H-1000 medium (Vector). A Zeiss LSM510 confocal microscope or a Leica inverted fluorescence microscope connected to a digital video camera (Leica DC100) were used to obtain confocal or fluorescence images. Confocal image analysis was performed using the LSM 5 Image Browser (program version 2.8). 4–6 independent experiments were performed and the final data presented selected as the most representative of the results obtained. Immuno-Electron (EM) and Reflection Contrast Microscopy (RCM) After treatment with IGF1, cells were fixed for 15 minutes in 2% paraformaldehyde/0.1% glutaraldehyde and spun down at 7500 rpm for 5 min. Cells were embedded in 12% gelatine, spun PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e19846