Molecules and Cells

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Fig. 5.

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Fig. 5. (A) To quantify the overlapping region of cells between ST5 and Src, BMMs were overexpressed by a ST5 expression vector tagged with HA and treated with M-CSF (30 ng/ml) and RANKL (150 ng/ml) for 2 days. Cells were subjected to immunofluorescence. The fluorescence intensity of Cy3 (anti-Src)- and FITC (anti-HA)-labeled cells was analyzed by confocal microscope. The images of four microscopic fields per sample were randomly captured. The overlapped coefficient values of Cy3 and FITC among 32 cells per sample were determined by Zen 2009 software (Carl Zeiss). The experiments were performed in triplicate wells per group. Scale bars = 100 μm. (B and C) Transfected BMMs were starved for 5 h and treated with RANKL (500 ng/ml) for the indicated times. Protein levels of phospho-forms and total forms of Src and Syk. (D and E) ST5-deficient cells were incubated for 2 days with M-CSF (30 ng/ml) and RANKL (150 ng/ml). (D) To measure Ca2+ oscillations in individual cells, cells were loaded with Fura-2/AM for 30 min and monitored by a confocal laser. Each colored line represents calcium oscillation in a cell. (E) BMMs transfected with siRNA were stimulated with M-CSF (30 ng/ml) and RANKL (150 ng/ml) for 3 days. The levels of nuclear and cytoplasmic NFATc1 proteins were assessed by Western blotting. *P < 0.05, ***P < 0.001 (by t-test).
Mol. Cells 2019;42:810~819
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