Anti flag antibody

Anti-FLAG antibody (Origene, Rockville, MD) was used to detect the FLAG-tagged DN-Tcf4. Actin was used a loading control using anti-actin antibody (Sigma, St. Louis, MO). Protein isolation and western blotting were performed as previously described [12 (link)-14 (link)].

HEK 293T cells expressing α_vβ₆ integrin (induced lentiviral expression as described above) were cultured in 100 mm dishes and incubated for 6 h at 37°C with Del-1 (1 μg/mL; R&D Systems) and/or LAP (0.5 μg/mL, R&D Systems). A previously described protocol was used (34 (link)), with some modifications. Briefly, the cells were washed with ice-cold PBS and lysed for 20 min in 900 μL of buffer containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 30 mM Tris-HCl, pH 7.5, 150 mM NaCl, and complete protease inhibitor cocktail, Next, the mixture was centrifuged at 19,000 × g for 15 min. Cell lysates were precleared for 2 h at 4°C with Protein G Mag Sepharose (GE Healthcare), incubated overnight at 4°C with an anti-Flag antibody (diluted 1:200; OriGene), and then incubated for 5 h with 25 μL of Protein G Mag Sepharose. Complexes containing antibody-bound Ag and coprecipitated proteins were pelleted and washed twice with 1% CHAPS buffer, and then three times with TBS. Bound proteins were eluted with 50 μL of 1× RIPA sample buffer and analyzed by western blotting with antibodies specific for Del-1 (Proteintech), LAP (Santa Cruz Biotechnology), Flag (OriGene), and β-actin (Cell Signaling Technology).

(2 × 10⁷) CD8⁺ T-cells isolated from peripheral lymph nodes using a CD8⁺ selection kit (EasySep) were activated with plate bound anti-CD3, CD28 overnight. CD8⁺ cells were then transfected with Flag-tagged Nr4a1 and cross-linked with 2 mM DSG (Sigma) and 1% formaldehyde methanol-free (Thermo Scientific Pierce). Cells were then lysed in RIPA buffer with protease inhibitors (Sigma), and chromatin was sheared by sonication in a Bioruptor sonicator (Diagenode). Immunoprecipitation was carried out with Dynabeads Protein A (Life Technologies) and Anti-Flag antibody (Origene). DNA was isolated using a ChIP DNA Clean and Concentrator column (Zymo). Target genes were amplified from the isolated DNA using SYBR® green master mixture (Roche). anti-Nur77 (M-210) and Anti-CoREST (Santa Cruz) was used for IP.