Measurement of Relative Rates of Leptin Biosynthesis桝fter preincubation of adipose tissue fragments in minimal essential medium (without methionine and cysteine, Sigma) in the absence (basal) or presence of insulin (6 nM) for 1 h at 37 癈, the media were exchanged for minimal essential medium containing [³⁵S]methionine/cysteine (500 礐i/ml, Easy-Tag^TM EXPRE³⁵S³⁵S protein labeling mix, PerkinElmer Life Sciences) under the same hormonal conditions, and the incubation proceeded for another 45 min for labeling. Preliminary experiments showed that the incorporation of [³⁵S]methionine/cysteine into total protein is linear up to 90 min and that [³⁵S]methionine/cysteine incorporation into leptin is linear up to 60 min in rat adipose tissue. [³⁵S]Leptin was then immunoprecipitated from tissue homogenates as described previously (12) using a polyclonal human leptin antibody from rabbit (Biovender, Brno, Czech Republic) followed by 4?2% SDS-PAGE. [³⁵S]Leptin bands were detected using PhosphorImager (Storm 860, GE Healthcare), and band intensities were quantified with ImageQuant software 5.0 (GE Healthcare). The completeness and specificity of immunoprecipitation was confirmed. Incorporation of the label into trichloroacetic acid-precipitable protein was determined (13), and rates of leptin biosynthesis under different conditions were normalized to this value to calculate relative rates of leptin biosynthesis. Lipoprotein lipase (LPL) was immunoprecipitated by adding 3 礸 of chicken anti-bovine LPL antibody (gift from Dr. J. Goers, California Polytechnic State University, San Luis Obispo, CA) as described previously (14).