Tamoxifen dosing and confirmation of knockout efficiency

To determine the dose of tamoxifen required for Cre activation, ROSA-YFP:K14CreER^+/− mice were injected intraperitoneally with tamoxifen (75 mg/kg dissolved in corn oil) for 5 consecutive days. After 48 h of the last injection, mice were sacrificed and checked for YFP expression by IFA in dorsal skin sections. For experimental purposes, both K14CreER^−/−Dab2^fl/fl and K14CreER^+/−Dab2^fl/fl (Dab2-cKO) were injected intraperitoneally with tamoxifen (75 mg/kg dissolved in corn oil) for 5 consecutive days. To check the Dab2 KO efficiency in the epidermis, mice were sacrificed at PD68, and dorsal skin sections were collected. We incubated the skin sections in 10 ml 0.25% trypsin O/N at 4 °C. The next day, 10 ml fresh trypsin was added followed by incubation at 37 °C for 30 min. The epidermis was then peeled off from the dermis using a surgical blade and further processed for single-cell suspension. The cells were lysed in trizol for RNA isolation.