Fig 2: (A) Schematic representation of the p27-SCF-Skp2 complex. (B) Format of the HTRF assay to detect Skp2-Cks1 interactions. The Skp1/Skp2 complex with GST tags and Cks1 with His6 tags bind to the anti-GST-Eu donor beads and the anti- His6-d2 receptor beads, respectively. Donor and receptor beads are close to each other by GST-Skp1/Skp2 binding to His6-Cks1. When excited at a wavelength of 337 nm, the donor bead produces energy, one part is released at a wavelength of 620 nm, and the other part is transferred to the receptor bead by energy resonance and released at a wavelength of 665 nm. When inhibitors inhibit Skp2 binding to Cks1, only energy is released at a wavelength of 620 nm. (C) Purified gel and molecular sieve diagrams of protein GST-Skp1/Skp2. (D) Purified gel and molecular sieve diagrams of protein His6-Cks1. lane 9: Cut the GST-Tag by thrombin, lane 10–14: Final purification protein of His6-Cks1.

Fig 3: The binding experiment of GST-Skp2 and His6-Cks1 in vitro. (A) The pull-down experiment verified whether the protein was active in vitro: lane1: GST-Skp2; lane2: His6-Cks1; lane3: GST-Beads + GST-Skp2; lane4: GST-Beads + His6-Cks1; lane5: GST-Beads + GST-Skp2 + His6-Cks1; lane6: His-Beads + GST-Skp2; lane7: His-Beads + His6-Cks1; lane8: His-Beads + GST-Skp2 + His6-Cks1. (B–D) The protein optimal concentration of GST-Skp2 binding to His6-Cks1 was determined by orthogonal experiment, Skp2: 30 nM, Cks1: 20 nM.

Fig 4: Skp2-2 inhibits cell cycle progression in SGC-7901. (A,B) SGC-7901 cells treated with increasing concentrations of Skp2-2 for 48 h. (C) (D) SGC-7901 cells treated with 50 μM Skp2-2 for 0–48 h. Fixed cells were collected and the cell cycle was measured by flow cytometry. (E,F) SGC-7901 cells treated with 50 μM Skp2-2 for 4 h, the stability of protein Skp2 was measured at different temperatures. The representative data of at least three independent experiments are shown, *P < 0.05, **P < 0.01. compared with the control group (mean ± SD, n = 3).

Fig 5: Effect of exogenous expression of p62-HA in p62 knockdown C13 cells. (A) Western blot analysis using anti-Skp2, anti-p62/SQSTM1, anti-p27, anti-p21 and anti-HSP90. (B,C) Real time PCR analysis of Skp2 and p62 mRNA expression in NTC and p62 (sh280) C13 cells upon QC treatment. GAPDH was used as an internal control in the mRNA analysis experiments.