To detect cellular level of target proteins, protein extracted from THCA cells were detected by Western Blot. Lysis buffer consisting of Tris (50 mM, pH7.4), EDTA (1 mM), NaCl (150 mM), 10% Glycerol, 1% Triton, as well as protease/phosphatase inhibitor cocktail (Roche, Basel, Switzerland) was used to extract cell lysates. Bradford assay was performed to determine protein content. Later, we performed SDS-PAGE to separate 30 to 40 μg soluble proteins. Afterwards, electrophoretic transfer of the isolated proteins to PVDF membranes (Millipore, Billerica, MA, USA) was conducted. Primary antibodies utilized in the present study were diluted into 5% nonfat milk as 1:500. Immunoreactive proteins were visualized using EasyBlot ECL kit (Sangon Biotech, China). Gray value of each protein band of western blots picture were analyzed by ImageJ (Rawak Software, Inc. Germany).

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