Expression of CavY14F mutant decreases IGF-IR internalization in IGF1 stimulated cells.

<p>(A) Hacat cells were transiently transfected with pEGFPN1 Cav-1-wt and pEGFN1 Cav-1Y14F plasmids. 48 hours after transfection serum starved HaCat cells were stimulated with IGF1 10 nM for 5 min, fixed in 4% formaldehyde, permeabilized with Methanol at −20°C, labeled with a rabbit anti-PTRF/Cavin (red) and a mouse anti-IGF-IR (blue) imaged by confocal immunofluorescence microscopy. Merged fields show co-localization (white) of Cav-1wt, PTRF/Cavin, IGF-IR (upper pannels) and Cav-1Y14F, PTRF/Cavin and IGF-IR (bottom panels). (B) 48 hours from the transfection, serum-starved cells were treated with IGF110 nM for the indicated times, trypsined, washed, blocked and incubated with a mouse PE-conjugated IGF-IR.antibody. PE-conjugated IGF-IR labeled cells were analyzed by flow-cytometry to measure plasma membrane IGF-IR expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014157#s3" target="_blank">Materials and Methods</a>. Data are expressed as the mean ± SD. Statistical analysis was performed using Student's <i>t</i> test. *p<0.05.