The mouse skin tissue was collected from fourteen (14) day-old mice. The lysate was prepared by homogenization in modified RIPA buffer (50 mM Tris -HCl, pH 7.4, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% sodium dodecylsulfate (SDS), 1 mM sodium ethylenediaminetetraacetate, 1 mM phenylmethylsulfonyl flouride, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin). Tissue and cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris -HCl pH 6.8, 12.5% glycerol, 1% SDS, 0.01% bromophenol blue) containing 5% b-mercaptoethanol.

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