Gtx101087

The inhibition of VHL–HIF-1α interactions was investigated using a co-IP assay following the manufacturer’s instructions. Briefly, HEK293 cells (1 × 10⁶ cells/well) were treated with indicated concentrations of complex 1a, P1 (30 μM) or DMSO for 2 h. After cell lysis and protein lysate separation, 100 μg of total protein was incubated with VHL antibody (1:1000; GTX101087, GeneTex) at 4 °C overnight. The proteins were immunoprecipitated using agarose beads. The levels of co-precipitated HIF-1α-OH were visualized using ECL Western Blotting Detection Reagent (GE Healthcare).

In IHC, paraffin blocks of the xenograft tumors were cut into 4-μm sections. The slides were incubated with primary antibodies: IGFBP3 (1:200, MAB305; R&D Systems, Minneapolis, MN, USA), HIF-1α (1:500, H1alpha67-ChIP Grade; Abcam, Cambridge, UK), HIF-2α (1:1,000, ab8365; Abcam, Cambridge, UK), HO-1 (1:200, GTX101147, Genetex, Irvine, CA, USA), or Von Hippel-Lindau (VHL) (1:200, GTX101087, Genetex, Irvine, CA, USA) at 4 °C overnight (about 17 h).
In ICC, 0.2 × 10⁵ cells were cultured on 25 mm × 25 mm glass slides and incubated under hypoxic or normoxic conditions for 17 h. Then, the slides were incubated with primary antibodies: IGFBP3 (1:200; MAB305; R&D Systems, Minneapolis, MN, USA), HIF-1α (1:300; GTX127309; Genetex, Irvine, CA, USA), or HIF-2α (1:300; GTX30114; Genetex, Irvine, CA, USA) at 4 °C overnight (about 17 h).
A chromogen in IHC and ICC was developed, following the protocol of UltraVision Quanto Detection System HRP DAB (Thermo Fisher Scientific, Waltham, MA, USA). The sections or slides were stained with hematoxylin for examination, and photos were taken using Moticam X3 Plus (Motic, Speed Fair Co., Ltd, HK) microscope camera by Motic Images Plus 3.0 software. The signals of IHC and ICC were quantified and analyzed by ImageJ 1.53 k software (NIH, USA).