P50 sc 8414

The whole cell lysate was prepared in a lysis buffer with sonication as described elsewhere²¹ (link). In IP, the lysate protein (500 μg at 1 μg/μL) was mixed with 2 μg antibody in 100 μL protein G agarose (16–266, Millipore, Billerica, MA, USA) and rotated overnight at 4 °C. The agarose beads were washed with 800 μL washing buffer three times and collected as IP product after centrifugation at 13,000×g for 30 s. The succinylation signal was detected in the IP product after resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Antibodies included: HDAC1 (H6287, Sigma), histone deacetylase 3 (HDAC3, ab7030; Abcam, Cambridge, UK), sirtuin 1 (SIRT1, ab12193; Abcam), SCD1 (ab19862, Abcam), SREBP1 (sc-8984, Santa Cruz, CA, USA), pan anti-succinyllysine polyclonal (106,768, NovoPro, shanghai, China), PPARγ (made in our laboratory), P50 (sc-8414, Santa Cruz, CA, USA), and β-actin (sc-47778, Santa Cruz, CA, USA).