Anti flag epitope

Nuclear extracts were derived from cell lines in midlog phase growth at maximally 70% confluence [20 (link)]. Protein concentrations were determined (Bio-Rad Protein Assay Dye Reagent Concentrate; Bio-Rad, Marnes-la-Coquette, France #5000006). Then, 30 µg of nuclear extracts was separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman Protran BA85; Sigma-Aldrich, Saint-Quentin-Fallavier, France #WHA10402525) as described [10 (link)]. Antibodies employed: anti-POLR3G (Santa Cruz Biotechnology, Dallas, TX, USA; sc-28712), anti-POLR3GL (Atlas Antibodies, Bromma, Sweden; HPA027288), anti-beta-actin (Santa Cruz Biotechnology; sc-81178), anti-FOXA1 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-101058), anti-AR (Santa Cruz Biotechnology, Dallas, TX, USA; sc-7305), anti-Flag epitope (Sigma Aldrich, Burlington, MA, USA; F1804). R.G. Roeder kindly provided antibodies directed against RPC39 and RPC62. Proteins were visualized using clarity^TM Western ECL (Bio-Rad, Marnes-la-Coquette, France #1705060). Beta-actin served as loading control.

Specific antibodies were purchased from the following commercial sources: anti-Flag epitope and anti-β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-c-Fos and anti-NFATc1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Recombinant human soluble RANKL and human M-CSF were previously described [31 (link),32 (link)]. RAW264.7 and PlatE cells were cultured in Dulbecco’s modified Eagle’s medium (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) and an antibiotic/antimycotic solution (Welgene) at 37 °C in a 5% CO₂ atmosphere [31 (link)]. C57BL6/J mice were purchased from Daehan Biolink (Umsung, Korea) and maintained under pathogen-free conditions. Pax5^Tg mice expressing the human Pax5 transgene under the control of the TRAP promoter were generated by the Transgenic Core Facility of the Korea Advanced Institute of Science and Technology (Daejeon, Korea). The transgenic mice were backcrossed onto the C57BL6/J background for five generations. The genotype of Pax5^Tg mice was analyzed by PCR with the following primers: Pax5^Tg (sense), 5′–CCC TGC TGA CAT CGG GAG CAG T–3′; Pax5^Tg (antisense) 5′–GTC GTC ATC GTC TTT GTA GTC C–3′. All animal work was approved (approval no. CNU-00114, 1 January 2018) by the Animal Experiment Ethics Committee of Chungnam National University.