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Combinatorial omics analysis reveals perturbed lysosomal homeostasis in collagen VII-deficient keratinocytes

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Academic year: 2021

Partager "Combinatorial omics analysis reveals perturbed lysosomal homeostasis in collagen VII-deficient keratinocytes"

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Published in "Molecular & Cellular Proteomics doi: 10.1074/mcp.RA117.000437, 2018"

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