Antibodies against flag

Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested with nocodazole (15 µg/ml) for 2 hours at 30°C. Samples were cross-linked with 1% formaldehyde for 20 min at room temperature, and cross-lining was stopped with 0.125M glycine. Chromatin was sonicated using a Bioruptor sonicator bath (Diagenode), to an average fragment size of 250 bp. Soluble chromatin was immunoprecipitated as previously described [18] (link) using antibodies against Flag (Sigma). Immunoprecipitation efficiency was checked by Western blot. Immunoprecipitated and input DNA were amplified using whole-genome amplification (Genome-Plex, Sigma-Aldrich).

ChIP assays were performed using the Magna ChIP A/G kit (Merck Millipore, Billerica, MA, USA) in accordance with the manufacturer’s instructions. MV4-11 cells with ectopically expressed flag-FEV were cross-linked in 1% formaldehyde and then sonicated to create soluble chromatin. Antibodies against flag (Sigma–Aldrich, St. Louis, MO, USA) were added to precipitate the DNA fragments. The recovered DNA was amplified using PCR or quantitative PCR.

Information of antibodies can be found in Supplementary Table S2 online. Antibodies against FLAG, β-actin, and the B2 subunit of V-ATPase were purchased from Sigma-Aldrich. Antibodies to V5, Ccz1, CD68, and the A subunit of V-ATPase were obtained from Thermo Scientific, Santa Cruz, Hycult Biotech, and Abcam, respectively. Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgGs were from GE Healthcare. Clean-Blot, an HRP-conjugated antibody for post-immunoprecipitation western blot detection of native primary antibodies, was purchased from Thermo Scientific. Alexa-conjugated secondary antibodies were from Thermo Scientific.

The anti-RHBDD1 mouse monoclonal antibody was prepared in-house. Antibodies against Na⁺/K⁺-ATPase, TACE, phospho-c-Raf, c-Raf, phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2, phospho-EGFR, EGFR and α-tubulin were purchased from Cell Signaling Technology. Antibodies against FLAG, Myc, V5 and GST were purchased from Sigma-Aldrich. proTGFα, β-actin, GAPDH and GFP antibodies were purchased from Santa Cruz Biotechnology, Inc.

Vector containing Gentisate 1,2-dioxygenase (GDO) was a generous gift from Professor J. Kaplan (University of Utah, Salt Lake City, UT, USA). The plasmid was transformed into yeast cells harboring the pUG23, pUG23-CsMIT1 or pUG23-CsMIT2 plasmids, and transformants carrying both vectors were selected on SC/Glu-Ura-His solid media. Cells were grown in SC/Glu-Ura-His liquid medium overnight to mid-log phase and then for 12 h in fresh medium supplemented with 100 µM BPS. Cell lysates were prepared using glass bead homogenization, as described previously [57 (link)]. The presence of c-GDO in the yeast extracts was confirmed by Western blot analysis using antibodies against FLAG (1:10,000; Sigma Aldrich, St. Louis, MO, USA). The activity of the c-GDO enzyme was measured spectrophotometrically at 340 nm, as described previously [57 (link)], and was calculated using an extinction coefficient of 10.2 cm⁻¹ mm⁻¹. c-GDO activity was expressed as nmol of substrate converted per minute per mg of protein.