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Fig. 5. Biochemical fractionation and in situ nuclear-matrix extraction of HaCaT cells. (A) Western blotting. HaCaT cells grown in low-Ca²⁺ medium were subjected to a sequential extraction by CSK 0.1% Triton X-100 (C/T, 0.1%), CSK 0.5% Triton X-100 (C/T, 0.5%), chromatin digestion by DNase I (DNAse) and a final extraction by 0.25 M ammonium sulfate. Whole-cell protein extracts (P1) were prepared and, following each step, insoluble fractions P2, P3, P4 and P5 were pelleted, and soluble fractions S2, S3, S4 and S5 were retained for immunoblotting with monoclonal anti-CASK antibody and JoL2 antibody to detect lamin A/C. (B) HaCaT cells were grown on glass coverslips in low-Ca²⁺ medium and then subjected to in situ nuclear-matrix extraction using five sequential treatments with CSK buffer only (no extraction; stage I), CSK 0.1% Triton X-100 (stage II), CSK 0.5% Triton X-100 (stage III), DNase I digestion (stage IV) and 0.25 M ammonium sulfate extraction (stage V), and processed for indirect immunofluorescence microscopy using antibodies against CASK (green channel) and lamin A/C (red channel). (C,D) HaCaT cells grown in low-Ca²⁺ DMEM were transfected with a full-length CASK-GFP construct and subjected to in situ nuclear-matrix extraction using sequential treatments up to the indicated stages. GFP-CASK is shown in the green channel and lamin A/C in the red channel (C). Stages IV and V extracted cell showing colocalisation (arrowheads) of the nucleolus marker, nucleolin (red channel) with GFP-CASK (green channel) (D). Scale bars, 50 µm (B), 20 µm (C,D).

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