2013
08/06

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Generic Peptides

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Figure options To examine the effect of GRX

2.2. Cell culture and GRX1 overexpression H9c2 cells, a clonal line derived from embryonic rat heart cells, were obtained from the American Type Culture Collection (CRL-1446). The overexpression of GRX1 in H9c2 cells was accomplished using previously established methods [9]. Briefly, H9c2 cells were transfected with vectors that constitutively overexpress FLAG-tagged mouse GRX1 protein, using the vector pTRE2Hyg-GRX [9]. We successfully obtained three clones, and one of these clones (H9c2-GRX30) was used in this study. Mock-transfected H9c2 cells were used as a control (H9c2-Vector). H9c2-GRX30 cells had greater thioltransferase activity than either the parent H9c2 cells or the H9c2-Vector cells (data not shown) [9]. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) that had been supplemented with 10% fetal calf serum (FCS), 75 μg/ml G418, and 75 μg/ml hygromycin B in a humidified depsipeptide of 95% air and 5% CO2 at 37 °C. 2.3. Immunoblot analysis Cultured cells were harvested and lysed in lysis buffer (20 mM Tris–HCl [pH 7.2], 130 mM NaCl, and 1% Nonidet P-40 that included protease inhibitors [20 μM phenylmethylsulfonyl fluoride, 50 μM pepstatin, and 50 μM leupeptin]). Protein samples were electrophoresed on 10% or 12.5% SDS–polyacrylamide gels (SDS–PAGE) under reducing and non-reducing conditions and then transferred to nitrocellulose membranes using previously established methods [16]. The membranes were blocked with 5% skim milk in Tris-buffered saline (TBS; 10 mM Tris–HCl [pH 7.2] and 150 mM NaCl) and then incubated at 4 °C overnight with the primary antibody in TBS that contained 0.1% Tween 20. The blots were coupled with the peroxidase-conjugated secondary antibodies, washed, and then developed using a commercially available ECL detection kit (GE Healthcare, Little Chalfont, UK) in accordance with the manufacturer’s instructions.

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