Dinonyl phthalate

Adipocytes and SVF were isolated from SAT (n = 3, cohort 3) for analysis of ESR2 protein expression, as described previously [22 (link)]. Half a gram (500 mg) of adipose tissue was digested as described in section 2.3. The floating adipocytes were washed four times in Hank’s medium (6 mmol/L glucose, 4% BSA, 0.15 μmol/L adenosine, pH 7.4) and were separated from the medium by filtration through dinonyl phthalate (Merck, Darmstadt, Germany). The SVF was isolated from the collagenase medium by centrifugation for 3 min at 1200 rpm and washed with PBS. The adipocytes and SVF were used for immunoblotting analysis of ESR2, as reported in section 2.9. Total protein levels of the adipocyte and SVF fractions were measured using BCA protein assay kit (Pierce, Thermo Scientific) and used to extrapolate the total ESR2 protein levels in the whole-cell extractions.

Adipocytes and SVF were isolated from SAT (n = 3) for analysis of OCT3 protein, as described previously (13 (link), 14 (link)). Adipose tissue (500 mg) was digested using collagenase A (Roche) in a shaking water bath at 105 rpm for 1 hour at 37 °C in Medium 199 (Gibco, Life Technologies) supplemented with 6mM glucose, 4% bovine serum albumin (BSA, Sigma), 150nM adenosine (Sigma), pH = 7.4. The floating adipocytes were washed 4 times in Hank's medium (6 mmol/L glucose, 4% BSA, 0.15 μmol/L adenosine, pH = 7.4) and were separated from the medium by filtration through dinonyl phthalate (Merck, Darmstadt, Germany). The SVF fraction was isolated from the collagenase medium by centrifugation for 3 minutes at 1200 rpm and washed with phosphate-buffered saline (PBS). The adipocytes and SVF were used for immunoblotting analysis of OCT3. Total protein levels of the adipocyte and SVF were measured using the BCA protein assay kit (Pierce, Thermo Scientific) and used to extrapolate the total OCT3 protein levels in the whole tissue sample.