Figure 1.

Level of interaction between HMGCS2 and PPARα increases in a palmitoyl-CoA concentration-dependent manner. A) In vitro transcribed and translated [³⁵S]HMGCS2 was incubated with 1 μg of GS/PPARα prebound to glutathione Sepharose beads or GST Sepharose alone as shown, followed by SDS-PAGE. Fluorogram of the dried gel representing 10% of the flow through (FT) or the bound protein (B) is shown. B) One milligram of purified HMGCS2-His6 was incubated with various concentrations of palmitoyl-CoA or palmitate or the combination of 30 min of pretreatment in 10 mM NEM followed by palmitoyl-CoA as shown for 30 min. One hundred nanograms of HMGCS2 (10% of the reaction) was incubated with immobilized GSTPPARα followed by SDS PAGE and Western blot analysis with anti-His5 antibody. C) One microgram of purified HMGCS2-His6 was incubated with 25 μM [¹²⁵I]-iodopalmitoyl-CoA (lanes 1 and 3) or 25 μM [¹²⁵I]-iodopalmitate (lanes 2 and 4) following a 30-min preincubation with buffer alone (lanes 1 and 2) or 10 mM NEM (lanes 3 and 4). Ten percent of these reactions (100 ng protein) were incubated with immobilized GST-PPARα, followed by SDS PAGE gel. Autoradiography of the dried gels from the bound fractions and 5% of eluted fractions is shown.