4.11. Immunofluorescence Analysis

After treatment with heteronemin (0.62 μg/mL), 2% paraformaldehyde was used to fix cells for 30 min and 0.2% Trition X-100 in PBS was used for the permeabilization for 20 min [54]. 5% BSA and 0.05% Trition X-100 (T-PBS) were used to prevent non-specific protein binding for 1 h. Cells were then incubated with the phosphorylated talin and actin (1:250) for 2 h and the secondary antibodies for 1 h for 1:1000 (Alexa Fluor 586-conjugated goat anti-mouse IgG or Alexa Fluor 488-conjugated goat anti-rabbit IgG, Life Technologies, Carlsbad, CA, USA). After washing with PBS, cells were monitored with FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).