Pierce anti dykddddk magnetic agarose beads

We followed the protocol that we used recently.⁴⁵ (link) The plasmids of Lenti-FLAG-BRD4-GFP, Lenti-HA-EED, Lenti-FLAG-EED (and FLAG-tagged truncated EED constructs), Lenti-HA-STAT3-GFP, and Lenti-HA-EZH2-GFP were constructed and each was used to transfect HEK293 cells (Addgene, catalog no. 60360) in the presence of the JetPRIME transfection reagent. Cells were lysed on ice for 30 min in Pierce IP Lysis Buffer (Thermo Fisher Scientific, catalog no. 87788) containing Halt Protease Inhibitor Cocktail (87785; Thermo Fisher Scientific), and then centrifuged at 13,200 × g for 15 min at 4°C. The supernatant was incubated with 50 μL of Pierce Anti-DYKDDDDK Magnetic Agarose beads (A36797; Thermo Fisher Scientific) at 4°C overnight. The beads were washed 3× with cold PBS buffer and then incubated in 0.1 M glycine (pH 2.8) for 10 min at room temperature with frequent vortex to elute the immunoprecipitates. The eluate was neutralized with 1 M Tris-HCl, pH 8.5 (15 μL per 100 μL of eluate) and briefly heated at 95°C before its use for SDS-PAGE and western blot analysis.

pEGFP-N1-FLAG (empty vector) (60360; Addgene) and pFLAG-SREBP2Nt (N-terminal transcriptionally active domain, amino acids 1–482) (26807; Addgene) were used to transfect cells (HEK293). For co-IP, cells were lysed on ice for 30 min in Pierce IP Lysis Buffer (87788; Thermo Fisher Scientific) containing Halt Protease Inhibitor Cocktail (87785; Thermo Fisher Scientific), and then centrifuged at 13,200g for 15 min at 4°C. The supernatant was incubated with 50 μl of Pierce Anti-DYKDDDDK Magnetic Agarose beads (A36797; Thermo Fisher Scientific) at 4°C overnight. The beads were washed 3× with cold PBS buffer and then incubated in 0.1 M glycine (pH 2.8) for 10 min at room temperature with frequent vortex to elute the immunoprecipitates. The eluate was neutralized with 1M Tris–HCl, pH 8.5 (15 μl per 100 μl eluate), and briefly heated at 95°C before its use for SDS–PAGE and Western blot analysis.

HEK293T cells were transfected with 6 µg DNA (Flag-PRMT6-pcDNA3.1, PPARγ1-pcDNA3, PPARγ2-pcDNA3, pcDNA3 empty vector). Whole cell extracts were prepared using lysis buffer [50 mM Tris–HCl (pH 7.5), 150 mM sodium chloride, 10 mM sodium fluoride, 20 mM ß-glycerophosphate, 1% IGEPAL and 1 mM EDTA supplemented with protease inhibitor cocktail (Carl Roth, Karlsruhe, Germany) and 100 mM sodium orthovanadate. After incubation for 10 min, 7.5 mM Magnesium Chloride and 50 U Benzonase were added. The lysate was then incubated on a rotating wheel for 2 h at 4 °C followed by centrifugation at 14.000 rpm for 10 min at 4 °C. Protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Feldkirchen, Germany). 600 µg whole cell lysate and 50 µl Pierce Anti-DYKDDDDK magnetic Agarose-Beads (Thermo Fisher Scientific: Waltham, Massachusetts, USA) were used per IP. The reactions were incubated for 2 h on a rotating wheel at 4°C. The beads were washed twice with lysis buffer and subsequently boiled in 2 × Laemmli buffer for 5 min. The supernatant was applied to a 10% SDS-PAGE and analyzed by immunoblotting.