Total protein was extracted by cell lysis buffer supplemented with protease inhibitor. Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in TBS-T, membranes were incubated with the primary antibody (#ab129067, CTR1, Abcam, San Francisco, CA, USA). Protein was detected with Image Acquisition using ImageQuant LAS 4000 (Pittsburg, PA, USA).
For immunofluorescence assays, cells were cultured on glass slides with cell abundance of 30% in 6-well polystyrene microplates. The cells on glass slides were fixed with 4% formaldehyde and then permeabilized with PBS containing 0.2% Triton X-100 after washing three times in PBST. After blocking with 1% BSA in PBS, the cells were incubated with primary antibody (#sc-66847, CTR1, Santa Cruz, CA, USA) overnight at 4 °C. Cells were washed three times in PBS, followed by incubation with 2 μg/mL Alexa Fluor 594 phalloidin (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Cells were washed with PBS three times, nuclear stained with 5 μg/mL DAPI, and analyzed with a confocal laser scanning microscope (Olympus FluoView FV1000 confocal microscope, Melville, NY, USA).
For immunofluorescence assays, cells were cultured on glass slides with cell abundance of 30% in 6-well polystyrene microplates. The cells on glass slides were fixed with 4% formaldehyde and then permeabilized with PBS containing 0.2% Triton X-100 after washing three times in PBST. After blocking with 1% BSA in PBS, the cells were incubated with primary antibody (#sc-66847, CTR1, Santa Cruz, CA, USA) overnight at 4 °C. Cells were washed three times in PBS, followed by incubation with 2 μg/mL Alexa Fluor 594 phalloidin (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Cells were washed with PBS three times, nuclear stained with 5 μg/mL DAPI, and analyzed with a confocal laser scanning microscope (Olympus FluoView FV1000 confocal microscope, Melville, NY, USA).