Ab95950

Tissues or cells were lysed on ice in 1× RIPA lysis and extraction buffer (cat. #89900, Thermo Fisher Scientific) with Halt protease inhibitor (cat. #78441, Thermo Fisher Scientific). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (cat. #23225, Thermo Fisher Scientific). Total protein (20 µg) was loaded onto the gels, and blots were developed with primary antibodies against PHD3 (ab184714, Abcam, Cambridge, UK; 1:2000 dilution); HIF1α (D2U3T, Cell Signaling Technology, Danvers, MA, USA; 1:1000 dilution); HIF2α (D9E3, Cell Signaling Technology; 1:1000 dilution); fumarate hydratase (ab95950, Abcam; 1:3000 dilution); EGFR (sc-373746, Santa Cruz Biotechnology, Dallas, TX; 1:1000 dilution). pEGFR (sc-57545, Santa Cruz Biotechnology; 1:200 dilution); glutaminase (GLS1; ab93434, Abcam; 1:1000 dilution), glutaminase 2 (GLS2; NBPI-76544, Novus Biologicals, Littleton, CO, USA; 1:1000 dilution); alanine-serine-cysteine transporter 2 (ASCT2; ABN73, Millipore Sigma, 1:1000 dilution), and β-actin-horseradish peroxidase (5125S or 12262S, Cell Signaling Technology; 1:5000 dilution). Image J (National Institutes of Health, Bethesda, MD, USA) or Image Studio Lite (version 5.2, LI-COR Biosciences, Lincoln, NE, USA) were used to perform densitometry.

Immunoblotting was performed as previously described.^{11 (link),20 (link)} Briefly, MSC were plated
at 5 × 10⁴ cells per well in a six-well plate prior to lysis with
universal lysis buffer (Millipore). Protein quantification was performed with
Qubit Fluorometer and Quant-iT™ protein assay kit (Invitrogen) to ensure equal
loading of samples. Protein lysates were diluted 1:1 with 2 × Laemmli buffer and
denatured at 95°C, before loading on Tris HCl 4–20% ready gels (Bio-Rad). Gels
were transferred to nitrocellulose membrane and subsequently blocked with 5%
bovine serum albumin (Sigma) or 5% milk in tris-buffered saline–Tween for 1
hour. Incubation with primary antibody anti-FH (Abcam, ab95950),
anti-nuclear-related (erythroid-derived 2) factor 2 (anti-NRF-2) antibody
(R&D, AF3925), anti-hypoxia inducible factor1α (anti-hypoxia inducible
factor1α (anti-HIF-1α; Abcam, ab51608)), anti-COX5 (Santa Cruz, SC-376907) and
anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam, ab9484) was
performed overnight at 4°C. Amersham ECL Plus™ Western Blotting Detection System
(GE Healthcare) was used to visualise specific protein expression patterns by
chemiluminescence. The integrated density of bands was measured using ImageJ
(National Institute of Health, NIH), and values are expressed relative to GAPDH
loading control protein.

Immunostaining for Fh1, Sdhb and 2SC was performed on FFPE sections. The evaluation was performed by two pathologists in a single-blind manner (HHY and RRH). We used a commercially available primary rabbit anti-Human FH polyclonal antibody (1:800 dilution, Abcam ab95950), rabbit anti-Human SDHB (1:250 dilution, Sigma Aldrich HPA002868) and rabbit anti-Human 2SC polyclonal antibody (1:1000 dilution, Discovery Antibodies, crb2005017e) with a modified Agilent FLEX Envision detection system to run all steps in Automated Autostainer AS48Link. All FFPE slides were pretreated with Heat Induced Epitope Retrieval (HIER) in Dako PTLink using Envision FLEX Target Retrieval solution—Low pH (6.0) for FH, SDHB and high pH (9.0) for 2SC—and incubated 97°C for 20 minutes. Fh1 staining in allograft cells was considered negative in the presence of an internal positive control in immune or stromal cells or non-tumoral kidney parenchyma. 2SC staining was considered positive when there was 2SC-negative staining in the adjacent non-neoplastic cells. Flex Rabbit Negative Control Immunoglobulin fraction staining, instead of primary antibodies to Fh1 and 2SC, was also performed in parallel with experimental slides for each run as a staining negative control, for which all staining came out all clearly negative.