Sc 8031

Immunohistochemistry analysis was performed as previously described with minor modifications [52 (link)]. Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at 5 µm thickness, deparaffinized, and rehydrated. Antigen were unmasked by heat retrieval with pH 9 EDTA buffer for 15 min and endogenous peroxidase activity was inactivated with a 3% hydrogen peroxide solution for 10 min. Tissues were then immuno-stained overnight at 4 °C with anti-nucleolin antibody (sc-8031, Santa-Cruz Biotechnology, Heidelberg, Germany, 1:50) and diluted in antibody diluent (Zytomed, Berlin, Germany). Immuno-complexes were revealed using the anti-mouse Polink HRP mouse kit and the DAB substrate (Diagomics, Blagnac, France) with an incubation of 30 min at room temperature. Tissues were then stained with hematoxylin and dehydrated. Slides were mounted using Eukitt medium. Protein expression was scored as null (0), weak (1), moderate (2), and strong (3), and the percentage of tumor cells stained was noted. The multiplication of the score and the percentage of stained tumor cells gave the quick score (between 0 and 300). Analysis was performed separately by one genitourinary pathologist (MN) and by two scientific investigators (DD and VF).

MΦ were collected in lysis buffer (containing 6.65 M urea, 10% glycerol, 1% SDS, 10 mM Tris pH 6.8; pH 7.4) and incubated for 5 min at 95 °C with 1 × SDS sample buffer (5 × buffer consists of 100 mM Tris-HCl pH 6.8, 5% SDS, 25% glycerol, 0.01% bromphenol blue, 100 mM dithiothreitol) and resolved on polyacrylamide gels followed by transfer onto nitrocellulose membranes. Nonspecific binding was blocked with 5% milk powder in Tris buffer saline with 0.05% Tween-20 for 1 h at room temperature, followed by incubation with primary antibodies against PXN (Abcam, Cambridge, UK; #ab32084; 1:3000 dilution), TNS3 (ThermoFisher Scientific #PA5-63112; 1:750 dilution), and nucleolin (Santa Cruz Biotechnology, Heidelberg, Germany; #sc-8031; 1;3000 dilution) according to the manufacturer’s instructions. Proteins were visualized by IRDye secondary antibodies using the Li-Cor Odyssey imaging system (all from LICOR Bioscience, Bad Homburg, Germany). Uncropped blots are presented in Supplementary Figures.

Rabbit antibodies against NCL (ab22758) and HMGB1 (ab67281) and a mouse TLR2-blocking antibody (ab9100) were obtained from Abcam (Cambridge, UK). A mouse anti-NCL antibody (sc-8031) was purchased from Santa Cruz Biotechnology, Inc (Dallas, TX). Lipopolysaccharide (LPS) and mouse IgG1 (M9269) were from Sigma-Aldrich (St. Louis, MO). Recombinant human TLR2-10xHis (2616-TR) was from R&D Systems (Mineapolis, MN). Anti-HA-agarose was from ThermoFisher Scientific (Walthem, MA). A TLR4-blocking mouse antibody (Mabg-htlr4), a TLR5-blocking human antibody (Maba-htlr5), an interleukin (IL)-1β-blocking mouse antibody, lipoteichoic acid (LTA, tlrl-slta), flagellin (tlrl-stfla), and poly I:C (tlrl-picw), were from InvivoGen (San Diego, CA).

We ran our samples on 8.7% and 12% SDS PAGE and transferred them to a nitrocellulose membrane as described before [34 (link)]. We performed WB analyses using anti-NCL antibodies (C23, cat # Sc-13,057, and sc-8031 Santa Cruz; ABIN183989 by antibodies-online GmbH, and ZN004 mouse monoclonal antibody, cat# 39–6400 ThermoFisher Scientific) and visualized our product with horseradish peroxidase-conjugated to affinity-purified goat anti-rabbit IgG and goat anti-mouse (1:5000 dilution; H + L; Jackson ImmunoResearch); anti-hnRNP G (kindly provided by Prof. Stefan Stamm, University of Kentucky, Lexington); anti – YWHAQ (Thermo Scientific) visualized with horseradish peroxidase-conjugated to affinity-pure goat anti-rabbit IgG (H + L; Jackson Immunoreaserch, 1:5000), and with anti-Sm, as described [34 (link)].