Horseradish peroxidase conjugated immunoglobulin g igg

Cells were homogenized and lysed using radioimmunoprecipitation assay lysis buffer (100 mM NaCl; 50 mM Tris-HCl, pH 7.5; 1% Triton X-100; 1 mM EDTA; 10 mM β-glycerophosphate; 2 mM sodium vanadate; and protease inhibitor). Protein concentrations were detected by using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Forty micrograms of protein was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane using a commercial semidry blotting apparatus (Bio-Rad, Richmond, CA, USA). The immunoblot was incubated for 1 h with blocking solution (5% skim milk) at room temperature. Next, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-CUL4B (1:500 dilution), anti-E-cadherin (1:500 dilution), anti-N-cadherin (1:1,000 dilution), anti-vimentin (1:500 dilution), anti-β-catenin (1:1,000 dilution), anti-c-Myc (1:500 dilution), and anti-cyclin D1 (1:200 dilution) (all Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with TBS-T, the membranes were incubated at room temperature for 1 h with a 1:2,000 dilution of horseradish peroxidase-conjugated immunoglobulin G (IgG) (Santa Cruz Biotechnology). Target protein was detected by an enhanced chemiluminescence substrate kit (Pierce Biotechnology, Rockford, IL, USA).

The human SSC line treated with JAZF1-siRNAs or control siRNA and miRNA-31-5p mimics or miRNA mimics control were lysed with RIPA buffer (DingGuoChangSheng Biotech, Bengjing, China) containing protease inhibitor cocktail (MedChemExpress, USA). After 30 min lysis on ice, cell lysates were cleared by centrifugation at 12,000 × g, and the concentrations of proteins were measured by Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Forty micrograms of cell lysate from each sample were used for SDS-PAGE (Bio-Rad Laboratories, Richmond, CA), and Western blots were performed according to the protocol as described previously. The chosen antibodies included PCNA (Abcam), JAZF1 (Sigma-Aldrich, USA), cyclin A2 (Santa Cruz), cyclin D1 (Santa Cruz), cyclin E1 (Santa Cruz), and ACTB (Proteintech). After extensive washes, the membranes were incubated with horseradish peroxidase-conjugated immunoglobulin G (IgG) (Santa Cruz Biotechnology) at 1:2,000 dilution for 1 hr at room temperature. The blots were detected by Chemi-Doc XRS system (Bio-Rad Laboratories, Hercules, CA, USA), and densitometric analyses were processed with ImageJ software. The relative levels of proteins were normalized to the expression of ACTB.