P mlc2 antibody

HUVECs were plated on coverslips in six-well plates and transfected with si-CCM1 or si-CCM2 for 24 hours. Cells were treated with ponatinib (1 μM) for a further 24 hours before fixation and immunocytochemistry staining with p-MLC2 antibody (Cell Signaling Technology). Images were taken with a Zeiss LSM800 confocal microscope.

For immunocytochemistry, 30,000 cells were seeded in a four-well chamber (Thermo Fisher Scientific) and treated with glucose as described above. After fixation with 4% paraformaldehyde for 15 min at room temperature, cells were blocked with blocking buffer (Cell Signaling Technology) for 60 min at room temperature. Then cells were incubated with p-MLC2 antibody (Cell Signaling Technology, 3671) diluted 1:50 overnight at 4°C. Secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes, R37116) was used for visualization. For costaining with filamentous actin (F-actin), cells were stained with Acti-stain 555 phalloidin (Cytoskeleton, PHDH1) as described in the manufacturer’s instructions. Then DAPI (Invitrogen, R73606) was added for nucleus staining. Confocal microscopy images were acquired using LSM900 scanning microscope (Carl Zeiss) equipped with 63×, 40×, and 20× magnification objectives. To determine subcellular localization of pMLC2 and F-actin, confocal microscopy images were analyzed with line profiles using ZEN 3.4 software (Carl Zeiss). The colocalization of pMLC2 and F-actin signals was evaluated with Pearson’s correlation coefficient (r).