Ab59149

Cells (HPDE6-C7, CaPAN-1, BxPC-3, PANC-1, and AsPC1) were lysed via RIPA buffer (Beyotime, Shanghai, China) and protein was collected. Concentrations of total protein were quantified through a BCA Protein Assay Kit (Sigma-Aldrich). Equal amounts of each sample were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transmembrane to polyvinylidene fluoride (PVDF) membranes (Merck, Darmstadt, Germany). Immediately, the membranes were incubated with primary antibodies overnight at 4°C, which were FBXL7 (ab59149, Abcam; 1.25 μg/ml), E-Cadherin (ab231303, Abcam; 1 μg/ml), N-cadherin (ab18203, Abcam; 1 μg/ml), Vimentin (ab137321, Abcam; 1:3,000), Snail 1 (ab53519, Abcam; 3 μg/ml), Snail 2 (ab82846, Abcam; 1:500), ZEB 1 (ab155249, Abcam; 1:3,000), Twist (ab49254, Abcam; 2.5 μg/ml), and GAPDH (ab181602, Abcam; 1:10,000). Goat anti-Human IgG/HRP (Solarbio, SE101) was employed as secondary antibody to incubate the membranes. Finally, Positive signals were detected with an ECL luminescence reagent (Sangon Biotech, Shanghai, China).

293T, human NSCLC cell lines containing H1975, HCC827, and A549, and normal non-tumor cells HBE, these cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Following ATCC protocols, all cells were maintained in a humidified incubator (37°C, 5% CO₂) and underwent mycoplasma analysis every two months. Chemicals, incorporating SDS, NaCl, Tris base, DMSO, and lipofectamine™ 2000 (#11668019), were gained through Sigma-Aldrich (St. Louis, MO). Dioscin, cycloheximide (CHX) and MG132 were sourced from Selleck Chemicals (Houston, TX). The RPMI-1640 and DMEM culture media, along with Fetal Bovine Serum (FBS) and penicillin-streptomycin (P/S), were sourced from Invitrogen (Grand Island, NY) to support the cell culture process. Primary antibodies targeting ki67 (#ab16667, IHC: 1:300) and FbxL7 (#ab59149, IB: 1:1000) were obtained from Abcam (Cambridge, United Kingdom) for use in this study, cytochrome C (#11940, IB: 1:1000), survivin (#2808, IB: 1:1000, IP: 1:200, IHC: 1:200), ubiquitin (#3936, IB: 1:1000), β-actin (#3700, IB: 1:10000), α-tubulin (#3873, IB: 1:5000), VDAC1 (#4661, IB: 1:2000), Bax (#14796, IB: 1:1000), cleaved-caspase 3 (#9664, IB: 1:1000, IF: 1:400) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). We obtained the Flag-survivin construct from Origene (RC205935).

IHC staining of PCa and matched tumor-adjacent tissues with antibodies against FBXL7 (ab59149, Abcam) were carried out according to the manufacturer’s declaration. In brief, the tissue specimens were fixed using formalin (10%) and embedded in paraffin. Serial sections of 5-μm thickness were sliced from the paraffin-embedded blocks. The slices were washed with TBS solution containing 0.025% Triton x-100 twice, and gently stir for 5 min each time. Then, TBS solution containing 10% normal serum and 1% BSA was used to seal at room temperature for 2 h. After incubating with FBXL7 antibody (8 μg/ml) overnight at 4°C, the sections were incubated with a Goat Anti-Rabbit IgG H&L (HRP) (1:5,000, ab205718, Abcam) at room temperature for 1 h. The sections used DAB-chromogen for development. Immunostained slides were photographed via a microscope and measured by Image J software.