Image lab analysis software 3

Cells were lysed in cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). The protein concentration was determined using the Bicinchoninic Acid Protein Assay Kit (Pierce Biotechnology, Inc., Thermo Fisher Scientific, Inc.). Protein was separated with 12% SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Life Technologies; Thermo Fisher Scientific, Inc.). The PVDF membrane was then blocked in 5% non-fat milk in PBS (Life Technologies; Thermo Fisher Scientific, Inc.) containing 0.1% Tween-20 (Sigma-Aldrich, Inc.) at room temperature for 3 h. Subsequently, the PVDF membrane was incubated with rabbit anti-human polyclonal LASP1 (1:100, Abcam, Cambridge, MA, USA) or rabbit anti-human GAPDH (1:100, Abcam) primary antibodies for 3 h at room temperature. After washed with PBS for 10 min, the PVDF membrane was incubated with goat anti-rabbit secondary antibody (1:10000, Abcam) at room temperature for 1 h. After washed with PBS for 10 min, the protein bands were detected using the Enhanced Chemiluminescence Western Blotting Kit (Pierce Biotechnology, Inc.; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols, and then quantified using Image Lab analysis software 3.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was used as the internal reference.