Gtx30114

Cell sample harvesting and immunoblot analysis were performed as presented previously [50 (link)]. To detect proteins, the primary antibodies β-actin (sc-47778, Santa Cruz Biotechnology, USA), vinculin (Sigma-Aldrich), HIF-1α (GTX127309, Genetex, USA), HIF-2α (NB100–122, Novus Biologicals, USA or GTX30114, Genetex, USA), p70 s6 kinase and phospho-p70 s6 kinase (Thr 389) (9202 and 9205 respectively, both from Cell Signaling, USA), and MF20 (a hybridoma producing both antibody detecting α and β MHC isoforms; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) were used in 5% non-fat milk/TBS-T at 4°C overnight. Corresponding secondary HRP-conjugated anti-rabbit or anti-mouse antibodies (in 5% non-fat milk/TBS-T, 1 h, RT; Cell Signaling, USA) were employed. Immunoreactive bands were detected using ECL detection reagent kit (Pierce, USA) and exposed to radiographic film (AGFA, Belgium). The adjustment of brightness and contrast was applied to the whole image.

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).