Pierce protein a g magnetic agarose

30 μL of resuspended Pierce protein A/G magnetic agarose (Thermo Scientific #78609) were washed 3X in dilution buffer (150 mM Tris 7.4, 100 mM NaCl, 10 mM EDTA), transferred to 1.0 mL of RIPA buffer with 15 μg ProteinTech 10782-2-AP, and incubated for 2 hours at 4°C with constant rotation. For optional crosslinking: beads were then washed 3X in 0.1% TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20), then 2X in PBS, before resuspension in 5 mM BS3 crosslinker in PBS (Thermo Scientific #21580). Crosslinking occurred for 30 minutes at room temperature with constant rotation, followed by quenching for 15 minutes with 1:10 of 1M Tris-HCl, pH 7.5. Non-crosslinked immunoglobulins were stripped by washing beads 3X in 1 mL Gentle Ab elution buffer (Thermo Scientific #21013). Beads were washed again 3x in TBS-T and then blocked overnight in Intercept TBS non-protein-based blocking buffer (Licor Biosciences #927-60001) with 1 mM DTT and 1% Tween-20 under constant rotation.

For immunoprecipitation (IP), HEK293TN were transfected with V5-TG2 and FLAG-Zbtb7a constructs using PolyJet transfection reagent (Signagen SL100688) following the manufacturer’s protocol. The cells were treated with VA4 12 hours after transfection and collected 48 hours after VA4 treatment. The cells were lysed and collected in IP lysis buffer (150mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40 in PBS). Protein concentrations were measured using a BCA assay. A 300 μg aliquot of lysate was immunoprecipitated with 4 μL of rabbit anti-V5 tag antibody (CST 13202S). After addition of primary antibody, the samples were incubated on a rotator at 4 °C overnight. IgG control samples were incubated with an equivalent amount of normal rabbit (Millipore 12–370) IgG antibody. After 18 hours, 30 μL of Pierce protein A/G magnetic agarose, (Thermo Scientific 78609) washed in IP wash buffer (2mM EDTA, 0.1% NP-40 in PBS) and blocked in 1% BSA in PBS, were added. After a 4 hour incubation, rotating at 4 °C, the samples were thoroughly washed in IP wash buffer and then in IP lysis buffer. After washing, beads were incubated in 30 μL of 2.5x SDS in IP lysis buffer for 10 min at 100 °C. Samples were then immunoblotted as described above. For quantification, ImageJ was used to measure the intensity of FLAG and V5 bands in each lane and the FLAG signal was normalized to that of V5.