After 36 h  of starvation (DMEM, 0.5% FBS, penicillin/streptomycin), quiescent HPASMCs were incubated with BMP2 for 10min or TGFB1 30min. ChIP was performed using the Low Cell# ChIP kit (C01010072; Diagenode) according to the manufacturers instructions with minor modifications described by Weiske and Huber (Weiske and Huber, 2006). First antibodies to the proteins of interest (PPARg (sc-7196X; Santa Cruz), Smad3 (9523; Cell signaling), Tata binding protein (sc-204X; Santa Cruz) and IgG (Diagenode)) were bound to magnetic beads. While binding took place, cells were fixed with 2mM disuccinimidyl glutarate (Sigma) for 45min at room temperature. Subsequently, the cells were washed twice with PBS. Chromatin was cross-linked for 7min at room temperature using 1% formaldehyde and washed twice with ice-cold PBS. The cross-linking reaction was stopped by incubation with glycine at a final concentration of 0.125M for 5min at room temperature. The cells were washed with ice-cold PBS and scraped from the cell culture dish in PBS and centrifuged. Each cell pellet was resuspended in a detergent-containing buffer, and chromatin was sheared by controlled sonification using the Covaris S2 sonicator. The sheared chromatin was divided into several aliquots depending on the number of antibodies which were used plus input control followed by incubation with the antibody-coated beads overnight. The beads with bound antibody-protein-DNA complexes were washed and dsDNA was collected using IPure kit (C03010014; Diagenode).
SUB2384842
AB-IgG_shPPARg-C_Control_1