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RT-PCR analysis; Total cellular

c-met and VE-cadherin protein levels vary in cultured HUVECs in relation to cell density. (A) HUVECs grown as a confluent monolayer or as a sparse monolayer were fixed and double-stained with anti–c-met C-28, and with anti–VE-cadherin antibodies, followed by immunofluorescent-labeled secondary antibodies. Original magnification for all images in panel A, × 400. In parallel, 5 μg protein extracted from confluent (c) or sparse (s) HUVECs was resolved by 8% SDS-PAGE and analyzed by Western blotting. Blots probed with anti–c-met C-28 (B), or with anti–VE-cadherin antibody (C) are shown, along with the corresponding scanning densitometry results. Arrows indicate the positions of the unprocessed 170-kDa c-met precursor and the 140-kDa c-met β-chain. In panels B-C, relative density is reported in arbitrary units. (D) Filters were stripped and reprobed with an antiactin antibody. Data are representative of 6 independent experiments.

In Western blots obtained with HUVEC extracts, the c-met protein presented a characteristic 2-band pattern, with the upper band corresponding to an unprocessed 170-kDa precursor and the lower band corresponding to the 140-kDa β-chainFurthermore, several bands of between 40 and 62 kDa were revealed with the anti–c-met C-28 antibody. These bands were shorter c-met fragments, as demonstrated by experiments in which the antibody was neutralized with a blocking peptide. They most likely were generated by limited proteolysis of the intact c-met protein during experimental manipulation. In fact, the amount of these fragments in the HUVEC extracts increased when collagenase (containing some contaminating enzyme activities) was used to recover the cells. Furthermore, these fragments could be produced from the full-length c-met protein by mild collagenase treatment of EDTA-recovered HUVECs, which generally contain very little of the low-molecular-weight c-met species