Fig. 2

Calpain 1 cleaves FICD fragments. a Human CD14⁺ cells were cultured with M-CSF (20 ng·mL⁻¹) for 8 h to induce early signals, and then DAPT (10 μmol·L⁻¹) was added. The cells were cultured for additional 2 days. Protein expression of c-FMS, the Na⁺/K⁺pump, Lamin B1, and α-tubulin were determined by immunoblot analysis. ME membrane extracts, CE cytoplasmic extracts, NE nuclear extracts. b Immunocytochemical analysis of DAPI and c-FMS. The right panel shows a merged image. Scale: 200×. c Cells were starved for 3h and then stimulated with M-CSF for the indicated times. d, e Cells were treated with imatinib (0.3 μmol·L⁻¹, d or a c-FMS-blocking antibody (5 μg·mL⁻¹, e prior to the addition of M-CSF. Protein expression of the FICD was measured by immunoblot analysis. Lamin B1 and α-tubulin were used as controls for the nuclear and cytoplasmic fractions, respectively. f Human CD14⁺ cells were cultured with M-CSF (20 ng·mL⁻¹) for 8h to induce early signals, and then MDL 28170 (5 μmol·L⁻¹) was added. The cells were cultured for additional 2 days. Immunoblot analysis with anti-c-FMS, Lamin B1, and α-tubulin antibodies. g, h Calpain 1, 5, and 6 were knocked down with siRNAs. The cells were cultured with M-CSF for 12 h. g Efficiency of Calpain 1, 5, and 6 knockdown. h Immunoblot analysis of c-FMS, α-tubulin, and Lamin B1. All data are shown as the mean ± SEM. *P < 0.05 by two-tailed, unpaired t-test (g). Representative results from at least three independent experiments are shown.