Specific hrp conjugated secondary antibodies

Cultured cells were lysised by RIPA buffer supplemented with complete protease inhibitor (Roche, Mannheim, Germany) and phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). Aliquots (20 μg) of total protein extracts were resolved on SDS-PAGE gels and transferred onto PVDF membranes. The membranes were then incubated with antibodies against GR (1:10000; Cell Signaling Technology, Danvers, MA, USA), Zif268 (1:500; Abcam, Cambridge, UK), SYN1 (1:200; Abcam, Cambridge, UK), or β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. Subsequently, the membranes were incubated with specific HRP-conjugated secondary antibodies (1:10,000; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C Signals were detected using western blot chemiluminescence HRP substrate (Takara, Kusatsu, Shiga, Japan). Image J software was used to analyze the result.

The cellular proteins were extracted from SW620, A549 and HepG2 cells with RIPA buffer containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA), and equal protein amounts (protein concentration of lysates measured by BCA assay, Thermo Fisher Scientific, Waltham, MA, USA) were subjected to SDS-PAGE. After electrophoresis proteins were transferred onto Trans Blot Turbo Mini PVDF membranes with Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA), blocked with 5% BSA and incubated with specific primary antibodies: mouse anti-human ABCB1 (clone F4, Sigma Aldrich, St. Louis, MO, USA). Specific HRP-conjugated secondary antibodies were used (Sigma Aldrich, St. Louis, MO, USA), and protein bands were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) employing Mini HD (Uvitec, Cambridge, UK). MDR1/ABCB1 Sf9 insect membranes (Solvo Biotechnology, Budaörs, Hungary) were used as a positive control for the presence of ABC proteins, and β-actin serves as an internal control.