Gel filtration molecular weight markers

Purified gp33¹⁰³ was dialyzed into a buffer containing 10 mM Tris, pH 8.0, 1 mM BME, 500 mM NaCl, and 4.5% glycerol. 1.2 mL of protein was run over a G-120 column using FPLC, 0.5 ml elution fractions were collected and peaks was identified with UV 280 absorbance measurements. Gel filtration molecular weight markers (Sigma-Aldrich) were resuspended in the same buffer at manufacturer recommended concentrations and run over the same column. The molecular mass for the gel filtration standards were plotted against elution volume (Ve) over void volume (Vo) to determine the molecular mass of gp33¹⁰³ based on its elution volume.

Immunoblotting was performed using a standard protocol. The redox state of TRX-1 was determined in native gels without additon of 2-β-mercaptoethanol in samples, as described previously17 . Thiol groups in cell lysates were firstly alkylated with iodoacetic acid (50 mM) in a 6 M guanidine-HCl buffer with 0.5% Triton X-100. For sucrose gradient separation, cell lysates were laid on the top of a four-layer sucrose gradient (10, 20, 40 and 60%). Samples were centrifuged at 35,000 g for 18 hours and fractions were submitted to polyacrilamide gel electrophoresis (SDS-PAGE). A mix of proteins from 12–200 kDa (Gel filtration molecular weight markers, Sigma-Aldrich) was used as control in sucrose grandient centrifugation, with the separated proteins stained with Coomassie Blue. For immunoprecipitation analyses, cell lysates were incubated with anti-TRX-1, protein A/G and agarose immunoprecipitation reagent (Santa Cruz), green fluorescent protein-(GFP)-Trap A (Chromo Tek GmbH, Munich, Germany) or anti- myc immunoprecipitation kit (Sigma-Aldrich, St. Louis, USA) as appropriate. Protein band densitometry was quantified by using Image J software.

H9c2 cells were scraped, transferred into tubes, and centrifuged at 2,800 g at 4°C for 5 min. The cell pellet was resuspended in 250 μl lysis buffer (50 mM Tris–HCl pH 7.2, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X‐100 containing protease cocktail and a proteasome inhibitor Mg132 2 μg/ml). Cell lysates were laid at the top of a sucrose gradient (10%, 20%, 40%, and 60%, top to bottom) prepared in 50 mM HEPES buffer pH 7.5, containing 100 mM KCl, 2 mM MgCl₂, 1 mM EGTA, and 1 mM EDTA. Samples were centrifuged at 35,000 g (4°C, 18 h). Fractions 1–16 (F1–F16) were collected from the base of the column. Each fraction was split into two 200 μl aliquots, one for immunoblotting and the other for immunoprecipitation experiments. As a control for density gradient separation, a mix of proteins (Gel filtration molecular weight markers, Sigma‐Aldrich) was added to the top of the sucrose gradient in a separate tube and centrifuged. The fractions obtained were submitted to SDS–PAGE, and proteins were stained with Coomassie Blue.