Anti skp1

For Western blot assay, cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (MedChemExpress, USA) on ice for 30 min. The protein concentrations were quantified by BCA assay. Subsequently, equal amounts of protein were extracted by 10% SDS-PAGE and transferred onto Immobilon®-P transfer membranes (Millipore, USA). Then, the membranes were blocked in 5% skim milk at 37 °C for 1 h and incubated with primary antibodies anti-SKP1 (1:1000, Cell Signaling Technology, USA), anti-cyclin D1 (1:1000, Cell Signaling Technology, USA), anti-p21 (1:1000, Cell Signaling Technology, USA), anti-N-cadherin (1:1000, BD Biosciences, USA), anti-occludin (1:1000, Boster, BioEngineering Company, Wuhan, China) and anti-GAPDH (1:5000, Proteintech, China) at 4 °C overnight. Next, the prepared membranes were incubated with secondary antibody (1:5000, Aksomics, China) at 37 °C for 1 h. Finally, the blots were visualized by ECL chemiluminescent reagent (Bio-Rad, USA). Western blot band intensity was analyzed by Image Lab™ 4.0 software and GAPDH was used as a loading control to normalize the amount of protein.

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).