Bicinchoninic acid assay kit

Cells in the logarithmic growth phase were seeded into 6-well plates with DEX, HQH or a co-treatment of DEX and HQH for 24 hours. Following treatment, cells were harvested, washed with PBS and lysed in lysis buffer (Beyotime Institute of Biotechnology). Bicinchoninic acid assay kit (Biosharp) was used to determine protein concentration following the manufacturer’s protocol. Samples were boiled for 10 minutes at 95°C and then allowed to cool to room temperature. The same amount of protein (30–50 μg/lane) was loaded on a 10% SDS-PAGE gel (Beyotime Institute of Biotechnology) and subsequently transferred to a PVDF membrane (EMD Millipore). Membranes were blocked in 5% nonfat milk and incubated at 4°C overnight with a primary antibody against GRα, GRβ, pERK, total ERK, BAX, Bcl-2, cleaved-caspase-3, β-actin or histone H3 (all used at 1: 1000 dilution), and subsequently incubated with the appropriate HRP-conjugated secondary antibody (1: 5000). Immunoreactive bands were visualized using ECL and imaged using a UVP Biospectrum 600 (UVP LLC). The loading control was β-actin.

Cells and xenograft tumor tissues were lysed by RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), and protein concentrations were measured by using the bicinchoninic acid Assay kit (BioSharp, Hefei, China). Protein samples (30–50 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), followed by blocking with Tris-buffered saline containing 0.2% Tween-20 and 5% fat-free milk at 37°C for 40 min. Subsequently, membranes were incubated with primary antibodies at 4°C overnight. After being washed three times, the biotinylated secondary antibody antibody-HRP (dilution, 1:1,000; cat. no., 00001-2; Wuhan Sanying Biotechnology Wuhan, China) was incubated for 1 h at room temperature and then washed again. At last the gray values were analyzed by using the Odyssey 3.0 software (https://odyssey-software1.software.informer.com/3.0).