Ab183039

For protein extraction, ground tissue or cells were lysed using RIPA lysis buffer, and the debris was removed by centrifuge. The protein in the supernatant was collected and quantified using a bicinchoninic acid assay (BCA) kit (Beyotime, China). The extracted proteins were separated on 12% SDS-PAGE gels, the voltage was first 80 V for ~30 min and subsequent 120 V, for ~60 min, and then and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) (400 mA, 90 min). The membranes were blocked using skimmed milk in Tris-buffered saline Tween 20 (TBST) and probed using primary antibodies for 12 h at 4°C. After washing; the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for another 1 h at room temperature. Immunoreactive bands were detected using chemiluminescence detection kits (Millipore).
The primary antibodies used were as follows: SKP2 (ab183039, 1:500; Abcam, UK), β-actin (ab6276, 1:3,000; Abcam), LC3B (ab192890, 1:2,000; Abcam), p62 (#5114, 1:1,000; CST), PHLPP1 (ab135957, 1:1,000; Abcam), β-tubulin (#2146, 1:1,000; CST), AKT (#4691, 1:1,000; CST), phosphorylated-AKT (#4060, 1:2,000; CST), mTOR (#2972, 1:1,000; CST), phosphorylated-mTOR (#5536, 1:1,000; CST). Antibodies against HA tag (ab18181, 1:2,000; Abcam), Myc tag (ab9106, 1:1,000; Abcam), Flag tag (ab125243, 1:2,000; Abcam).

Four micrometers thick paraffin-embedded sections of human tissues or xenografts were prepared. The sections were dewaxed, incubated with citric buffer at 95°C for 10 min, and blocked using H₂O₂ at room temperature for 20 min. The sections were incubated with normal goat serum for another 30 min at room temperature, followed by overnight incubation at 37°C with primary antibodies (SKP2, 1:100, ab183039 [Abcam]; Ki-67, 1:150, ab16667 [Abcam]). The sections were washed using PBS and incubated with HRP-conjugated-secondary antibody. For peroxidase detection, the sections were stained with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) for approximately 15 s and counterstained with hematoxylin.

PTC cells after indicated treatment were washed using PBS and fixed with 4% polyformaldehyde (PFA) for 20 min. The excess PFA was removed by washing; the cells were permeabilized using 0.1% Triton X-100 for 10 min and incubated with 5% normal goat serum for 60 min to prevent nonspecific binding. Cells were probed with primary antibodies at 4°C overnight, followed by incubation for 1 h with fluorescent secondary antibodies (1:200, Alexa-488-conjugated goat anti-rabbit IgG, ab150077; Abcam). The cells were counterstained using 4’,6-diamidino-2-phenylindole (DAPI; Sigma, USA) for 10 min at room temperature and visualized. The images were captured using a confocal microscope (Olympus, Japan). The primary antibodies used were anti-SKP2 (ab183039, 1:200; Abcam) and LC3B (ab192890, 1:100; Abcam).