Immobilon western chemiluminescent hrp reagents

The glioma cells transfected with siNC or STEAP3 siRNAs were lysed with RIPA buffer supplemented with protease inhibitor cocktails (B14012; Bimake, Houston, TX, USA). The BCA protein assay kit (23229; Thermo Fisher Scientific, Waltham, MA, USA) was applied to determine protein concentrations. Protein was transferred into 0.45 µm PVDF membranes (IPVH00010; Millipore, Burlington, MA, USA) after SDS-polyacrylamide gel electrophoresis. Then, the membranes were blocked with 5% nonfat dry milk for 1 h at room temperature. Primary antibodies against STEAP3 (PA5-20406; Thermo Fisher Scientific, Waltham, MA, USA) and β-actin (sc-58673; Santa, Dallas, TX, USA) was incubated overnight at 4 °C. The protein bands were visualized with Immobilon Western chemiluminescent HRP reagents (WBKLS0500; Millipore, Burlington, MA, USA).

To begin this procedure, cell lysates were processed with RIPA buffer supplemented with protease inhibitor. The total protein concentration was detected by BCA protein assay kit. Equivalent amounts of protein were loaded in each lane of SDS-polyacrylamide gel electrophoresis. Besides, the bands were transferred to PVDF membranes. Next, the membranes were blocked for 1 h at room temperature with TBST supplemented with 5% non-fat dry milk and then incubated at 4 °C overnight with primary antibodies. The primary antibodies displayed as follows: CDC42EP1 (1:500; Abcam, USA) and β-actin (1:5000; Santa Cruz, USA). Finally, protein bands were detected through the Immobilon Western Chemiluminescent HRP reagents (WBKLS0500; Millipore, USA).