Bloch and Richard T. Lee P. Christian Schulze, Heling Liu, Elizabeth ...

Schulze et al. NO regulates thioredoxin through Txnip-Supplemental Material - R1; page 4
Immunoprecipitation. Protein G sepharose beads (30 µl) were incubated with 1 µg anti-
Txnip antibody. Equal amounts of total protein lysates were incubated with antibody-bead
complexes for 2h rotating at 4°C. Beads were centrifuged and washed three times with 0.5 ml lysis
buffer and once with 0.5 ml ice-cold PBS. The beads were resuspended in SDS sample buffer,
incubated at 95°C for five minutes, and centrifuged. The supernatant was electrophoresed through a
SDS-PAGE system and signals visualized by enhanced chemiluminescence.
Thioredoxin Activity Assay. Thioredoxin activity was measured using the insulin disulfide
reduction assay as previously described. 4 Total cellular protein was extracted using lysis buffer, and
50 µg of cellular protein extracts were incubated at 37ºC for 15 min with 1 µL of activation buffer
(HEPES 50 mM, EDTA 1 mM, BSA 2 mg/mL, DTT 2 mM in water) in a total volume of 35 µL to
reduce thioredoxin. Reaction buffer (20 µL of HEPES 200 mM, EDTA 8 mM, NADPH 1.6 mg/mL,
insulin 5 mg/mL) was then added to the samples. The reaction was started by the addition of 5 µL
bovine thioredoxin reductase (American Diagnostica Inc., Greenwich, CT) or 5 µL water to control
cells, and the samples were incubated for 20 min at 37 ºC. The reaction was terminated by adding
250 µL of stop mix (guanidine chloride 6M, DTNB 400 µg/mL, Tris-HCl 200mM). Finally, the
absorption at 412 nm was measured spectroscopically. Thioredoxin activity was expressed per
milligram total protein.
Measurement of oxidative stress. Cells were incubated with 2’,7’-dichlorodihydrofluorecein
diacetate (DCFDA) for 45 min, washed in PBS and fluorescence intensity measured using a
fluorometer (Perkin Elmer) at 595 nm.
Statistical analysis. All experiments were performed at least three times and data are
expressed as mean ± s.d. The data were analyzed by Student’s t-test. One-way ANOVA with post-
hoc analysis was used for the analysis of data sets of more than two groups. P < 0.05 was
considered statistically significant.
Downloaded from
http://atvb.ahajournals.org/ by guest on July 30, 2013