Ecl western blot analysis detection system

Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF) (Millipore, USA). Blots were incubated with primary antibodies against SPHK1, GAPDH, phospho-ERK1/2, ERK1/2, phospho-AKT and AKT, followed by appropriate HRP-conjugated secondary antibodies. Immunocomplexes were visualized by ECL Western blot analysis detection system (Amersham Biosciences).

Total protein was extracted with radioimmunoprecipitation assay (RIPA) buffer containing 100 mmol of phenylmethylsulfonyl fluoride (Beyotime, China), and the protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Com Win Biotech, China). The primary antibodies were as follows: rabbit KLF3 polyclonal antibody (1:800, Abcam, USA), mouse PCNA monoclonal antibody (1:2,000, Santa Cruz Biotechnology, USA), mouse Bax monoclonal antibody (1:500, Santa Cruz Biotechnology, USA), and mouse β-actin antibody (1:1,000, Santa Cruz Biotechnology, USA). The protein bands were detected by an enhanced chemiluminescence (ECL) western blot analysis detection system (Amersham Biosciences, USA).