Keratinocyte Isolation and Lentiviral Transduction

Primary keratinocytes were isolated from WT CD-1 or TNR mice and cultured using previously described methods (Nowak and Fuchs, 2009 (link)). HEK293T cells were obtained from Takara Bio Inc. and used for lentiviral production as previously described. For lentiviral infections, keratinocytes were plated at 100,000 cells per six wells and incubated with lentivirus in the presence of 100 μl polybrene. 24–48 h after viral infection, cells were selected with 1–2 mg/ml puromycin. 96 h postinfection, keratinocytes were grown to confluency on fibronectin-coated coverslips and shifted to 2 mM Ca²⁺ growth media as previously described (Ezratty et al., 2011 (link)). Coverslips were fixed in either −20 Methanol (to visualize the microtubule cytoskeleton) or 4% PFA before processing for IF. Paraformaldehyde-fixed samples were permeabilized for 5 min in 0.5% Triton X-100. All antibodies were diluted in 10% normal goat serum in PBS.