All cell culture experiments were performed in accordance with institutional biosafety committee-approved protocols. HUVEC cells (Lonza, Allendale, NJ) were maintained in EGM Complete medium (Lonza, Cat# 3024A). Short-term murine endothelial progenitor cell cultures were developed from bone marrow of B6, BALB/C, or K10 knockout mice, following the protocol of Chunming Dong (25 (link)). Studies were performed on HUVEC and endothelial progenitor cells at 8 or fewer passages.
Cold exposure studies were performed in vitro by placing the cells in medium cooled to 4°C for the indicated time points (typically 1 minute for cold pre-exposure) before replacing the medium with 37°C medium.
Bacterial transfection and culture to express and purify K10 proteins was performed as previously described (20 (link),26 (link)). Plasmids expressing K10-MBP and hisK10 were obtained respectively from Thomas M. Magin and Jose L. Jorcano. Protein was purified from sonicated cell lysate by column chromatography (amylose or nickel columns), and following gel purification, was validated by MSMS and by immunoblot.
Cold exposure studies were performed in vitro by placing the cells in medium cooled to 4°C for the indicated time points (typically 1 minute for cold pre-exposure) before replacing the medium with 37°C medium.
Bacterial transfection and culture to express and purify K10 proteins was performed as previously described (20 (link),26 (link)). Plasmids expressing K10-MBP and hisK10 were obtained respectively from Thomas M. Magin and Jose L. Jorcano. Protein was purified from sonicated cell lysate by column chromatography (amylose or nickel columns), and following gel purification, was validated by MSMS and by immunoblot.
