Nb100 105

Protein extracts were homogenized and analyzed as previously described.²² (link) Briefly, an assessment of $\mathrm{HIF}\text{-}1\alpha$ protein content was accomplished using specific monoclonal antibody (NB100-105, Novus Biologicals, Littleton, Colorado) at a dilution of 1:1000 and appropriate horseradish peroxidase-linked secondary antibody alongside molecular weight ladder to verify size. Membranes were imaged on Protein Simple FluorChem M (Minneapolis, Minnesota) and analyzed using Image Studio Software (Li-Cor Biosciences, Lincoln, Nebraska). All blots were quantified within a linear range of exposure as previously optimized. All bands were normalized to the 45-kDa actin band of Ponceau S stain as a loading control. For each experiment, all groups were represented equally on each membrane and normalized to control condition.

The collected samples were lysed on ice with RIPA buffer which added phosphatase inhibitor and protease inhibitor. The protein lysates were isolated by SDS-PAGE and immunoblot analysis was performed. The antibodies were listed. Antibodies against AKT (4685, CST), p-AKT (4060 L, CST), CDCA8 (sc-376635, Santa Cruz), GSK3β (12456 S, CST), p-GSK3β (9323, CST), HIF1α (NB100-105, Novus Biologicals), PTEN (9559 S, CST), Snail (3879, CST), Slug (7585, CST), VEGFA (50661, CST), GAPDH (sc-365062, Santa Cruz), Flag tag (A4596, Sigma), HA tag (TA180128-1, OriGene), GFP tag (ab290, Abcam), Mouse-IgG (10283-1-AP, Proteintech), Rabbit-IgG (10284-1-AP, Proteintech), MK2206 (S1078, Selleck), were purchased from indicated companies.

Western blotting was performed as previously described^{22 (link), 23 (link), 46 (link)}. The lysates from the xenografts or cell lines with different treatments were homogenized in RIPA lysis buffer using a homogenizer. The supernatants were collected by centrifugation (12,000 rpm at 4 °C for 25 min; Beckman GS-6R). Protein was resolved by electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were probed with monoclonal HIF-1α (NB100–105, Novus biological, Littleton, CO, USA), Bax (2772 S, Cell signaling, Boston, MA, USA), Bcl-2 (3498S, Cell signaling), caspase-3 (9661S, Cell signaling), PARP (5625, Cell signaling), and PCNA (18197, Abcam, Cambridge, MA, USA), as well as p21 (Sc817, Santa Cruz, CA, USA), CDK4 (Sc70831, Santa Cruz), SP1 (ab27595, Abcam), HK2 (2772S, Cell signaling), PKM2 (4053S, Cell signaling), LDH-A (3582S, Cell signaling), and PDK1 (3820 S, Cell signaling). Following incubation with the primary antibody overnight at 4 °C, membranes were washed with Tris-buffered saline (pH 7.2) containing 0.05% Tween-20 and subsequently incubated with horse radish peroxidase (HRP)-conjugated anti-mouse (Sc-2005, Santa Cruz) or anti-rabbit (Sc-2357, Santa Cruz) secondary antibody for 1 h at room temperature. Bands of interest were analyzed using the ChemiDocTM Touch Imaging System (BIO-RAD, Hercules, CA, USA).

Formalin-fixed, paraffin-embedded specimens from a total of 30 primary ccRCCs (Supplementary Table 1) were retrieved from the archives of the Department of Pathology of the University of Heidelberg School of Medicine under Ethics committee vota 206/2005 and 207/2005, and informed consent by the patients. Sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Antigen retrieval was performed with a steam cooker using retrieval buffer (Target Retrieval Solution, Dako). Primary antibodies used were as follows: HIF-1α (Novus Biologicals, H1alpha67, NB100-105, 1:100), HIF-2α (Novus Biologicals, NB100-122, 1:100), phospho-mTOR S2448 (Cell Signaling, 49F9, #2976, 1:100), phospho-S6RP S235/236 (Cell Signaling, #2211, 1:50), Ki-67 (Dako, MIB-1, M7240, 1:100), CD31 (Dako, JC70A, M0823, 1:100) and CD45 (Dako, 2B11+PD7/26, M0701, 1:100). Immunodetection was performed using the Histostain-Plus Detection Kit (Invitrogen) according to the manufacturer's recommendations. The immunostaining for HIF-1α has been validated in a previous study using a ccRCC with a deletion in exon 2 of the VHL gene32 (link).

Hif1a^fl/fl or Hif1a^ΔSMC VSMCs treated with Ang II (1 μM) for 24 h were crosslinked in 1% formaldehyde in 1× PBS for 10 min. ChIP assays were performed for HIF1α (2 μg/IP, NB100-105; Novus Biologicals) or HIF2α (2 μg/IP, NB100-122; Novus Biologicals) using Simple ChIP Plus Kit (Cell Signaling Technology, Danvers, MA, USA) as previously described^{14 (link)}. The primers for ChIP assays are listed in Supplementary Table 1.

Cell samples were lysed in RIPA buffer on ice for 20 min. The lysed cells were collected and centrifuged at 14,000 x g to remove cell debris. Protein concentrations were determined with the BCA Protein Assay Kit (P0009, Beyotime). Each sample containing 30 μg of total protein was separated by SDS-PAGE in a 10% gel and transferred onto PVDF membranes (EMD Millipore Corporation, US). After blockage with 5% nonfat dry milk in Tris-buffered saline with 1‰ Tween (TBST), the membranes were incubated overnight at 4 °C with primary antibodies against brachyury (ab209665, Abcam), GLUT-1 (ab652, Abcam), SHH (sc-365,112, Santa Cruz), CD24 (ab64064, Abcam), KRT-8 (sc-8020, Santa Cruz), HIF-1α (NB100–105, Novus), NOTCH1 (4380, CST), JAGGED1 (ab109536, Abcam), HES1 (11,988, CST), and β-actin (CW0096S, CW Biotech). After three washes with TBST, the membrane was incubated with horseradish peroxidase–conjugated secondary antibodies (Jackson). The protein expression level was determined by densitometric analysis and was normalized to the level of β-actin.