Fig. 1. CLIC4 is upregulated in differentiating keratinocytes. (A) Newborn mouse and human foreskin keratinocytes were cultured in 0.05 mM Ca²⁺ medium for 4 days, then treated for 24 hours with the indicated concentrations of Ca²⁺ in the culture medium and lysates were examined by immunoblotting. (B) RNA isolated from mouse keratinocytes from A was amplified by RT-PCR, and CLIC4 and actin transcripts were detected by ethidium bromide staining of agarose gels. (C) Human foreskin keratinocytes were cultured in serum-free medium for 4 days and exposed to 5% FBS deleted of Ca²⁺ serum for the indicated times. (D) Mouse and human keratinocyte were treated with indicated doses of TPA for 24 hours, and lysates were analyzed by immunoblotting. The fold change for each band was determined by densitometry as described in Materials and Methods.