Figure 8.

Differentiation marker expression following Ca²⁺ treatment of stably transfected EMK cells expressing wild-type (SIK WT) or kinase-deficient Sik, where the conserved lysine in the catalytic domain has been changed to methionine (SIK 219K-M). Control cells contain the pLXSN retroviral expression vector alone (VECTOR). (A) Total protein was extracted from cells grown in low Ca²⁺ medium (0 h) or incubated in high Ca²⁺ medium for 14, 24, or 48 h. Immunoblotting was performed with keratin 1, loricrin, involucrin, or β-actin antibody. (B) Induction of transglutaminase mRNA expression during EMK cell differentiation. Total RNA extracted from cells grown in low Ca²⁺ medium (0) or in high Ca²⁺ medium for 14 or 24 h was hybridized with a probe for rat keratinocyte transglutaminase. As a control, the membrane was stripped and rehybridized with a probe for the ribosomal gene rpL32/4A (RPL32/4A) (31). (C) Increased filaggrin levels in differentiating cells overexpressing wild-type Sik. Equal amounts of total proteins from cells grown in low Ca²⁺ medium (0 h) or in high Ca²⁺ medium for 14 or 24 h were separated by SDS/PAGE, transferred to Immobilone-P membranes, and probed with anti-filaggrin antibody. As a control, the membrane was stripped and probed with β-actin antibody. Size markers (kDa) are at the left.