Five mg of lyophilised skin secretion was dissolved in 1 mL of mRNA stabilisation buffer and polyadenylated mRNA was isolated from this solution using magnetic oligo-dT beads as described by the manufacturer (Dynal Biotech, UK) and reverse-transcribed.
Total DNA was isolated from lymphocytes using buffer ATL, proteinase K, and RNase A (Qiagen, Inc., Valencia, CA) followed by phenol chloroform extraction and ethanol precipitation.
Protein was isolated from tissue using RIPA buffer (50 mg/ml) containing complete protease inhibitors (Roche).
Total protein was isolated from R3/1 cells using RIPA buffer and associated protease inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA).
Total protein was isolated from HT29 cells using 1%NP-40NP-40is buffer [150 mM NaCl, 10 mM HEPES (pH 7.45), 1% NP-40, 5 mM NaPyrophosphate, 5 mM NaF, 2 mM Na3VO4] containing protease inhibitor cocktail tablet (Roche, Mannheim, Germany).
Total RNA was isolated from cells and BAT fractions using Buffer RLT and purified by RNeasy Mini columns (QIAGEN).
Total protein was isolated from whole microdissected HFs using a specific buffer from the Human Phospho-Kinase ARY003BAR&D03B, R&D System), following the manufacturer's protocol.
DNA was isolated from the tissues incubated with the lysis buffer according to a standard protocol [ 13].
Genomic DNA was isolated from LV samples using the Genomic DNA Buffer Set and Anionic columns (Qiagen, Crawley, UK).
