Isotype control igg antibodies

ChIP assay was performed using ZymoSpin ChIP kit (Zymo Research, catalog # D5209) following the manufacturers’ guidelines. Briefly, HepG2 cells were seeded in 10 cm dishes and treated with either L165041 (20 μM) or DMSO (control) as described above. Following drug treatment for 24 h, cells were trypsinized, resuspended in PBS at a dilution of 8×10⁶ cells/mL and fixed with 1% formaldehyde (Sigma, catalog # 8775) for 10 min. Fixed cells were collected by centrifugation. To obtain nuclear lysates, fixed cells were resuspended in 500 μL chromatin shearing buffer and sonicated at 4°C in a Bioruptor 300 instrument (Diagenode, Inc.) for 12 cycles of 30sec ON: 30sec OFF at a “high” setting with intermittent vortexing. Chromatin containing nuclear lysates (100 μL) were incubated overnight with 4 μg of either rabbit anti-PPARδ (sc-7197x) or isotype control IgG antibodies (Santa Cruz Biotechnologies) and complexes were immunoprecipitated using protein-A magnetic beads. ChIP DNA was eluted, cross-linking was reversed and protein-free DNA was purified before PCR amplification with site specific primers covering the LDLR-PPRE region (Table 1).