Bradford protein assay kit

We used protein extracted from 129 human bladder tumors (57 non‐muscle‐invasive and 72 muscle‐invasive tumors) for RPPA analysis (Calderaro et al, 2014). The flash‐frozen tumor samples were stored at −80°C immediately after transurethral resection or cystectomy. All tumor samples contained more than 80% tumor cells, as assessed by the hematoxylin and eosin (H&E) staining of histological sections adjacent to the samples used for transcriptome analyses. All subjects provided informed consent, and the study was approved by the institutional review boards of the Henri Mondor, Foch and Institut Gustave Roussy Hospitals. RNA, DNA, and protein were extracted from the surgical samples by cesium chloride density centrifugation, as previously described (Calderaro et al, 2014). FGFR3 mutations were studied with the SNaPshot technique. The expression of FGFR3‐TACC3 and FGFR3‐BAIAP2L1 was analyzed by PCR, as previously described (Wu et al, 2013).
Lyophilized proteins were solubilized in Laemmli sample buffer and boiled for 10 min. Protein concentrations were determined with the Bio‐Rad Bradford Protein Assay Kit (Bio‐Rad, France).

U251 and LN229 cells were lysed using RIPA lysis buffer (Solarbio Life Sciences, Beijing, China) containing protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). After high-speed centrifugation (12000 rpm, 15 min, 4 °C), cell supernatants were collected. The concentration of protein in cell supernatants was measured using a Bio-Rad Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Next, protein (30 μg/sample) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were sequentially incubated with 5% non-fat milk for 1 h at room temperature, the primary antibody against ZNRF3 (cat. no. orb386705, Biorbyt, Cambridge, UK), IGF2BP3 (cat. no. 57145 S, Cell Signaling Technology, Danvers, MA, US), β-catenin (cat. no. 9582 S, Cell Signaling Technology), GAPDH (cat. no. 5174 T, Cell Signaling Technology), or Tubulin (cat. no. 5335 S, Cell Signaling Technology) for 12 h at 4 °C, and the corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Next, the membranes were exposed to the Pierce ECL Western Blotting Substrate (Thermo Scientific) to detect the protein signals.

We assessed expression of the TYRO3, AXL, MERTK and GAS6 genes by RT-qPCR, using 169 bladder tumour samples (87 NMIBCs and 82 MIBCs) from the previously described CIT-series cohort (“Carte d’Identité des Tumeurs” or “Tumour identity card”) of bladder tumours.^{5 (link),11 (link)} Seven normal urothelial samples were obtained from fresh urothelial cells scraped from the normal bladder wall and dissected from the lamina propria during organ procurement from a cadaveric donor for transplantation. RNA, DNA and protein were extracted from the surgical samples by cesium chloride density centrifugation, as previously described.^{5 (link),12 (link)} We used protein extracted from 21 human bladder tumours from the CIT-series (4 NMIBCs and 17 MIBCs) for western blot analysis.^{5 (link),12 (link)} Lyophilized proteins were solubilised in 1X Laemmli sample buffer and boiled for 10 min. Protein concentrations were determined with the BioRad Bradford Protein Assay Kit (BioRad, Marnes-la-Coquette, France) and TAM protein levels were assessed by immunoblotting.

Cell extracts were prepared as described previously [43 (link)]. Briefly, lyophilized proteins were solubilized in 1X Laemmli sample buffer and boiled for 10 min. Protein concentrations were determined with the Bio-Rad Bradford Protein Assay Kit (Bio-Rad, Marnes-la-Coquette, France) before immunoblotting. The following antibodies were purchased from Cell Signaling Technologies (Ozyme, Montigny-le Bretonneux, France): AXL (8661), ATM (2873), phospho-ATM (Ser1981)–(4526), ATR (2790), phospho-ATR (Ser428)–(2853), Chk1 (2360), phospho-Chk1 (Ser345)–(2348), Chk2 (2662), phospho-Chk2 (Thr68)–(2197), DNA-PKc (4602), MERTK (4319), TYRO3 (5585). Antibody against phospho-DNA PKc (Ser2056) (ab18192) was purchased from Abcam (Paris, France).

Cells were washed in PBS and lysed with RIPA buffer containing PMSP, DTT, and PI for 20 min on ice. The protein lysates were separated by centrifugation at 13,000 rpm for 20 min at 4 °C. The supernatant was transferred to a clean e-tube. Protein quantification was performed using a Bio-Rad Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Fifteen micrograms of protein was separated on 6–15% sodium dodecyl sulfate (SDS)-polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes (protran nitrocellulose membrane, Whatman, UK). Then, the membranes were blocked in PBS with 0.1% Tween-20 containing 1% BSA and 1.5% skim milk for 1 h. The membranes were incubated with primary antibodies at 4 °C overnight. Then, the membranes were incubated with HRP-conjugated secondary IgG antibodies (Calbiochem, San Diego, CA, USA) and probed using an enhanced chemiluminescence detection system (Amersham ECL kit, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA).
Antibodies against HIF-1α (#79233), HSP90 (#4877), vimentin (#5741), snail (#3879), and GAPDH (#5174) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MMP-9 (sc-21733) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

The following chemicals were used in the experiments; 5α-dihydrotestosterone (5α-DHT), methylglyoxal, 9,10-phenanthrenequinone and hydrindantin were purchased from Sigma (St. Louis, MO), and Ni-NTA chelating agarose CL-6B was purchased from Peptron company (Promega corporaton, USA). Bio-Rad Bradford Protein assay kit was purchased from (Bio-Rad Laboratories, Inc (South Korea). The others, including Na2HPO4, NaH2PO4, NaCl, bovine serum albumin (BSA), and imidazole, were purchased from Sigma (St. Louis, MO).