Ab68455

The cells were lysed and boiled at 96°C for 5 min, before loading onto four 20% SDS-polyacrylamide gels. Proteins were separated by electrophoresis in a Mini-PROTEAN Tetra cell chamber and transferred to polyvinylidene difluoride membranes. Then, the membranes were blocked in 5% non-fat milk (Yili Milk Company, China) in Tris-buffered saline-Tween (TBS-T) for 1 h at room temperature, and incubated with primary antibodies against Skp2 (ab68455, Abcam, UK), p27 (ab215434, Abcam), caspase-3 (ab2302, Abcam), and β-actin (ab8227, Abcam) overnight at 4°C with gentle shaking. Secondary antibodies conjugated to horseradish peroxidase (HRP) were applied for 1 h at room temperature, and immunoreactive bands were developed using enhanced chemiluminescence (Thermo Fisher Scientific, USA). The obtained bands were quantified in ImageJ x64 by normalizing to loading control and calculating band density relative to untreated control. Resulting graphs show an average of three independent donors.

IB assay was performed as previously described [27 (link)]. As to immunoprecipitation, cells were lysed, incubated with 2 μg corresponding antibodies overnight and incubated with protein A/G agarose for 4 h. The immunocomplexes were then resuspended with 2 × sample loading buffer and boiled for 5 min for SDS-PAGE and IB analysis. The primary antibodies used were: anti-Skp2 (ab68455, Abcam); anti-Skp1 (#12248, Cell Signaling Technology); anti-Cul1 (#4995, Cell Signaling Technology).