Figure 9.

Colocalization of CD44 and EGFR and impact of HA disruption. A: Western blot analysis of the association of CD44 and EFGR receptors. Cell protein was extracted from confluent monolayers of serum-deprived young dermal fibroblasts exposed to either serum-free medium alone (lane 1) or serum-free medium containing 10 ng/ml TGF-β1 in isolation (lane 2) or in combination with hyaluronidase (Hyal) (200 μg/ml) (lane 3) or 4MU (0.5 mmol/L) (lane 4) for 30 minutes. Subsequently samples were immunoprecipitated (IP) with anti-CD44 antibody, followed by immunoblotting with anti-EGF-R antibody. Specificity of immunoprecipitation was confirmed by negative control reactions performed with an IgG control (lane 5). B–Q: Immunohistochemical analysis of CD44 and EGF-R colocalization. Confluent monolayers of patient-matched young (B–I) and aged (J–Q) dermal fibroblasts were growth-arrested in serum-free medium for 48 hours, before addition of either serum-free medium alone (B–E and J–M) or serum-free medium containing 10 ng/ml TGF-β1 (F–I and N–Q). Cells were fixed and the expression of CD44 (C, G, K, and O) and EGF-R (D, H, L, and P) was examined by confocal section series using the Leica TCS SP2 AOBS confocal microscope, and their association was examined by merging of individual images (E, I, M, and Q). CD44 label is red, EGF-R is green, nuclei are counterstained blue with 4,6-diamidino-2-phenylindole (B, F, J, and N), and colocalization is represented by yellow. Arrows indicate colocalization at different magnification.