Fig. 2. CASK localisation is altered during keratinocyte differentiation. (A) HaCaT cells were grown in low-Ca²⁺ (0.06 mM) DMEM or in high-Ca²⁺ (1.6 mM) DMEM for 1 day (D1) and double stained with CASK (green channel) and a monoclonal antibody against E-cadherin (red channel) as indicated. (B) HaCaT cells were grown in low-Ca²⁺ or high-Ca²⁺ DMEM for 6 hours (6H), 1 (D1), 3 (D3) or 5 (D5) days and stained using a rabbit polyclonal antibody against CASK. Notice the gradual disappearance of nuclear CASK in cells grown in high-Ca²⁺ DMEM over time. (C) Nuclear (N) and cytosolic (C) extracts from HaCaT cells grown under low or high-Ca²⁺ conditions for 1 day (D1) and 5 days (D5), and immunoblotted using antibodies against CASK and -tubulin. (D) Human primary keratinocytes were grown in low-Ca²⁺ defined keratinocyte serum-free (SFM) medium or in high-Ca²⁺ SFM medium for 1 day (D1) or 3 days (D3) and stained with rabbit polyclonal antibody against CASK (green channel) and a monoclonal involucrin antibody (red channel). Scale bars, 50 µm.