Ab109617

Formalin-fixed, paraffin-embedded (FFPE) tissues were deparaffinized, and rehydrated in graded alcohols. IHC was carried out as previously described [37 (link), 38 (link), 60 ] using primary antibodies for PCNA (clone PC-10, Ab-1, Thermo Scientific, Waltham, MA, USA), KRT14 (NCL-LL002, Novocastra, Buffalo Grove, IL, USA), COX-2 (#12282, Cell Signaling, Danvers, MA, USA), S100A8 (T-1032, BMA, Augst, Switzerland), PDCD4 (LS-B1388, Lifespan Biosciences, Seattle, WA), STK40 (orb101780, Biorbyt, Cambridge, United Kingdom), FBXW7 (ab109617, Abcam, Cambridge, MA, USA), and PTEN (#9188, Cell Signaling) after citrate-based antigen retrieval. Protein was localized by incubation with 3-amino-9-ethylcarbazole substrate-chromogen (Dako, Carpinteria, CA, USA) or 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St Louis, MO, USA).

We performed western blotting as previously described39 (link). Cell lysates were prepared from BMECs transfected with agomiR-497∼195, antagomiR-497∼195, Fbxw7 siRNA, P4HTM siRNA or their negative controls in cell lyses buffer with 2% sodium dodecyl sulfate, 2 M urea, 10% glycerol, 10 Mm Tris-HCl (pH 6.8), 10 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. Total cell lysates were separated by SDS–polyacrylamide gel electrophoresis blotted on polyvinylidene difluoride membranes (Millipore). The membranes were incubated with specific antibodies to Fbxw7 (Abcam, ab109617, 1:1,000), P4HTM (Abcam, ab94626, 1:500), HIF-1α (Abcam, ab16066, 1:2,000) and NICD (Abcam, ab52301, 1:1,500) then reprobed with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, 1:10,000). Blots were developed using an ECL Kit (Santa Cruz), and exposed to x-ray films. The uncropped scans of blots are shown in Supplementary Figs 9 and 10.

Subsequent to protein extraction using protease inhibitor‐contained radio‐immunoprecipitation assay buffer (Boster, Wuhan, China), protein concentration was estimated using a bicinchoninic acid protein quantification kit (Boster). The protein sample was separated using freshly prepared 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis before transferring onto polyvinylidene fluoride membranes. Afterwards, overnight membrane incubation was conducted with primary antibodies (1:1000, Abcam) to rabbit anti‐FBXW7 (ab109617), rabbit anti‐c‐Myc (ab32072), rabbit anti‐Ki67 (ab197234), mouse anti‐HIF‐1α (Ab1), murine anti‐VEGF (ab1316) and murine anti‐GAPDH (ab8245) at 4ºC. Then, HRP‐labelled secondary goat anti‐rabbit (ab205719) or goat anti‐mouse (ab205719) antibodies (1:2000, Abcam) were added for 1‐h incubation at 37ºC. Electrogenerated chemiluminescence (ECL) working solution (EMD Millipore, Billerica, MA, USA) was supplemented to the member before 1‐min incubation at 37ºC. Subsequent to the removal of the excess ECL reagent, the blot was visualized by X‐ray in the dark for 5‐10 min and developed. The grey value of target protein bands was quantified using ImageJ software, with GAPDH utilized for normalization. Each experiment was repeated 3 times.

The paraffin-embedded tissue sections were deparaffinized and hydrated, followed by suppression of endogenous peroxidase activity by incubating in 0.3% H₂O₂ for 30 min at 37 °C. After PBS washing, the tissue sections were boiled in 10 mmol/L citrate buffer (pH 6.0) at 100 °C for 30 min, blocked with 5% normal goat serum at 37 °C for 1 h, and then incubated with FBW7 antibody (ab109617, 1:200, Abcam, Cambridge, UK) at 4 °C overnight. After that, the tissue sections were incubated with secondary antibody IgG (ab205718; 1:2000) at 37 °C for 1 h and then with horseradish peroxidase-conjugated streptavidin (1:1000 dilution) at 37 °C for 45 min, followed by treatment of newly prepared DAB for color development. All tissue sections were counterstained with hematoxylin. Finally, the stained tissue sections were analyzed under the OLYMPUS BX51 microscope.

Western blot analysis was performed as described before^{30 (link)}. The proteins were incubated with primary antibodies against FBXW7 (detecting all three isoforms, ab12292, abcam), FBXW7β (ab109617, abcam), cyclin E1 (#4129, CST), cyclin D1 (#55506, CST), CDK2 (#2546, CST), CDK4 (#12790, CST), CDK6 (#3136, CST), AKT (#4691, CST), p-AKT (#4060, CST), ERK (#4696, CST), p-ERK (#4370, CST), E-cadherin (#3195,CST), N-cadherin (ab18203), β-catenin (#8480), NF-κB p65 (#8242, CST), p-NF-κB p65 (#3033, CST), Snail (#3879, CST), Slug (#9585, CST), vimentin (#5741, CST), and β-actin (AP0733, Bioworld, China) at 4 °C overnight. The β-actin was regarded as the internal control.

Proteins were extracted using RIPA buffer (P0013B, Beyotime) containing a protease inhibitor and were quantified with a BCA kit (Thermo Fisher Scientific). Cell or tissue lysates containing equal amounts of protein were loaded in each well of a 10–17% SDS–PAGE gel. After electrophoresis, membrane transfer and blocking, membranes were incubated with the indicated primary antibodies and HRP-conjugated secondary antibodies (31430, 31460, Invitrogen). The bands were visualized by enhanced chemiluminescence using Clarity™ Western ECL Substrate (Bio-Rad). The following antibodies were used: anti-flag (1:5000, F1804, clone M2, Sigma-Aldrich), anti-EZH2 (1:1000, 07-689, Merck Millipore), anti-H3K27me3 (1:1,000, 9733S, clone C36B11, Cell Signaling Technology), anti-Histone H3 (1:2000, 4499S, clone D1H2, Cell Signaling Technology), anti-FBXW7 (1:1000, ab109617, Abcam), anti-DDX3 (1:1000, 11115-AP, Proteintech), anti-6xHis (1:1000, ab18184, clone HIS.H8, Abcam), anti-HA (1:1000, 35534, SAB), anti-β-tubulin (1:5000, T5201, clone TUB2.1, Sigma-Aldrich), anti-β-actin (1:5000, A1978, clone AC15, Sigma-Aldrich) and HRP-conjugated secondary antibodies, including anti-rabbit IgG (1: 10,000, 5220-0336, SeraCare), anti-mouse IgG (1:10,000, 5220-0341, SeraCare). A rabbit polyclonal antibody specific for EZH2-92aa (1:500) was produced by GenScript Biotech (Jiangsu, China).