Erythropoietin does not activate erythropoietin receptor signaling or lipolytic pathways in human subcutaneous white adipose tissue in vivo

Western blot analysis

Approximately 100 mg subcutaneous fat tissue was homogenized in homogenization buffer (Acute study: 20 mM HEPES, 10 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, 5 % SDS, 50 ?g/ml Soybean trypsin inhibitor, 4 ?g/ml Leupepsin, 0.1 mM Benzamidine, 2 ?g/ml Antipain, and 1 ?g/ml Pepstatin; Prolonged study: 50 mM HEPES, 20 mM NaF, 2 mM Na₃VO₄, 5 mM EDTA, 5 % SDS, HALT, 5 mM NAM, 10 ?M TSA) on a Precellys 24 (Bertin technologies, Montigny-le-Bretonneux, France). Hereafter, samples were thermo mixed at 37 °C and 500-1000 rpm for 1 h, followed by centrifugation at 14,000 x g for 20 min at room temperature. The homogenate was carefully separated from the lipid layer by a syringe, snap frozen, and centrifuged again, in order to purify the homogenate even further. The homogenate was frozen in liquid nitrogen and stored at -80 °C until further analysis.

In short, western blotting was performed as follows; 10 ?l homogenate was loaded onto a 4–15 % SDS gel (Criterion TGX stain-free gels, Bio-Rad, Hercules, CA, USA), followed by electro blotting onto a PVDF membrane. The stain-free technology was used to ensure equal loading [18]. Membranes were blocked with 2.5 % skimmed milk for 2 h before the primary antibody was added and incubated overnight at 4 °C. The following primary antibodies were used: From Cell signaling, Danvers, MA, USA; phospho-LYN (Thr507) (#2731), LYN (#2732), phospho-Akt (Ser473) (#9271), phospho-Akt (Thr308) (#9275), pan-Akt (#4691), phospho-p70S6k (Thr389) (#9205), p70S6k (#9202), phospho-STAT5 (Thr694) (#9359), STAT5 (#9358), phospho-p38MAPK (Thr180/Thr182) (#9211), p38MAPK (#9212), phospho-HSL (Ser660, corresponding to Ser650 in humans) (#4126), phospho-HSL (Ser563, corresponding to Ser552 in humans) (#4139), phospho-HSL (Ser565, corresponding to Ser554 in humans) (#4137), HSL (#4107), ATGL (#2138), HSP60 (#12165), SDHA (#11998), PDH (#3205), VDAC (#4661), phosphor-AMPK? (Thr172) (#2531), and PKA (#9624), from Abcam, Cambridge, UK; CGI-58 (#ab183739), anti-?-actin (#ab8227), and G0S2 (#ab80353), from Novus bio, Littleton, CO, USA; Cidea (#NB100-94219), from Abnova, Atlanta, GA, USA; Cidec (#H00063924-M07), from Millipore, Darmstadt, Germany; AMPK? pan (#07-181) and phospho-ACC (Ser79) (#07-303), from Amgen, Thousand Oaks, CA, USA; anti-Epo-R (#A82), from Southernbiotech, Birmingham, AL, USA: HRP streptavidin (#7100-05), from Santa Cruz, Dallas, TX, USA; G0S2 (#sc-133424), and from Pierce antibody production, Thermo scientific, Waltham, MA, USA; Perilipin (#PA1-1052). Following several washes, the membrane was incubated with the secondary antibody (donkey-anti-rabbit IgG, #NA934, Amersham, GE Healthcare, Pittsburgh, PA, USA/goat-anti-rabbit IgG, #sc-2054, Santa Cruz, Dallas, TX, USA) for 1 h at room temperature. Proteins were visualized by chemiluminescence detection system (Super signal dura extended duration substrate, Pierce, Thermo Scientific, Waltham, MA, USA/Clarity Western ECL substrate, Bio-Rad, Hercules, CA, USA #170-2054) using a ChemiDoc^TM MP imaging system (BioRad, Hercules, CA, USA). Precision Plus Protein All Blue Prestained Protein Standard (BioRad, Hercules, CA, USA #1610373) was used as molecular weight marker.