Mab 9165

Protein lysate from ANKRD1-overexpressing HEK293 cells was extracted via cell trypsinization, followed by gentle sonication and resuspension in ice-cold co-IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% Na-deoxycholate, 0.05% SDS, and protease inhibitor (Roche)Following centrifugation, the supernatant was thoroughly digested with DNase I (ThermoFisher) to remove any lingering cell debris. 250 ug of lysate was combined with 30 uL of V5-tag magnetic beads (M167-11; MBL) in 500 uL of co-IP buffer and incubated at 4 °C for 12 hours for the Co-IP. Lysate was also incubated with rabbit IgG (as a control) for 12 hours at 4 °C. The following day, IgG-lysate was incubated with protein A magnetic beads (ThermoFisher) for one hour. The beads were washed three times with 1 ml of co-IP buffer, resuspended in SDS-PAGE sample loading buffer, and heated at 98°Cfor 20 minutes. After samples were resolved on 8% SDS-PAGE, anti-ANKRD1 (dilution 1:200, sc-365056; Santa Cruz), anti-JUN (dilution 1:1000, mAb #9165; Cell Signaling), and anti-FOSL2 (dilution 1:1000, mAb #19967; Cell Signaling) antibodies were used for immunoblotting. Abcam’s VeriBlot IP Identification Reagent (dilution 1:1000, HRP; ab131366; secondary antibody) allows for the selective detection of target protein bands without background noise from denatured IgG heavy and light chains.

The in vitro protein interactions using recombinant proteins were performed as follows. GST-tagged ANKRD1 (100 ng), HIS-tagged JUN (truncated form 1-241aa, 100 ng), full-length JUN (100 ng), full-length FOS (100 ng), AP-1 DNA oligo (100 ng) (sc-2501, Santa Cruz), or T-5224 (20uM) (MedChemExpress) were mixed with Glutathione-conjugated beads in binding buffer (150 mM NaCl, 100 mM Tris ph8, 0.5% NP40, 10% Glycerol) for overnight (ON) at 4 °C on a rotating platform. After this time the complexes were washed 3 times in binding buffer, then resuspended in 30 µl of SDS-PAGE loading buffer for electrophoresis and heated at 95 °C for 10 minutes. We carried out the western blots by using anti -ANKRD1, -JUN, and -FOS antibodies (reported above) To determine the requirement of a native configuration the recombinant JUN was heated for 20 min in a thermal block at 98 °C, while the same amount of ANKRD1 or FOS proteins were left on ice as control, before mixing them to the pre-heated/denatured JUN protein. Western blot was carried-out using anti-ANKRD1 (sc-365056; Santa Cruz), anti-JUN (mAb #9165; Cell Signaling), and anti-FOS (mAb #2250, Cell Signaling). The concentration/dilution of each antibody is reported in Supplementary Data File 8.