Protein extracts were subjected to SDS-PAGE (6–12%) and transferred to a PVDF membrane (GE Healthcare, Hertfordshire, UK) as described [22 (link), 23 (link)]. The membrane was incubated with various antibodies as required at 4 °C overnight, followed by the addition of the corresponding secondary antibodies at room temperature for 1 to 2 h. An enhanced chemiluminescence (ECL) detection kit was used to detect antibody reactivity (Pierce, Rockford, IL, USA).
As described previously [25 (link), 26 (link)], 400 μg of nuclear protein was mixed with 8 μl of Protein A&G Sepharose (Sigma-Aldrich, Shanghai, China) and 8 μl of anti-LC3B antibody or immunoglobulin (IgG) control for 3 h at 4 °C. The protein-antibody complexes that were recovered on the beads were subjected to western blot analysis as above using anti-LC3B and anti-acetylated-lysine antibodies.
As described previously [25 (link), 26 (link)], 400 μg of nuclear protein was mixed with 8 μl of Protein A&G Sepharose (Sigma-Aldrich, Shanghai, China) and 8 μl of anti-LC3B antibody or immunoglobulin (IgG) control for 3 h at 4 °C. The protein-antibody complexes that were recovered on the beads were subjected to western blot analysis as above using anti-LC3B and anti-acetylated-lysine antibodies.
Free full text: Click here
