Proteins (50
μg) from all
considered conditions were treated as previously described.37 (link) Membranes were incubated for 12 h at 4 °C
with primary antibodies to Connexin-43 (1:500, Santa Cruz Biotechnology),
to Nkx 2.5 (1:500, Santa Cruz Biotechnology), and to β-actin
(1:1000, Santa Cruz Biotechnology), used as a housekeeping protocol.
Then, membranes were maintained at room temperature for 30 min with
a peroxidase-conjugated secondary antibody diluted 1:1000 in 1×
TBS, 5% milk, and 0.05% Tween-20. To visualize protein bands, the
ECL method was used; the protein level measurements were performed
by means of the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules,
CA).
μg) from all
considered conditions were treated as previously described.37 (link) Membranes were incubated for 12 h at 4 °C
with primary antibodies to Connexin-43 (1:500, Santa Cruz Biotechnology),
to Nkx 2.5 (1:500, Santa Cruz Biotechnology), and to β-actin
(1:1000, Santa Cruz Biotechnology), used as a housekeeping protocol.
Then, membranes were maintained at room temperature for 30 min with
a peroxidase-conjugated secondary antibody diluted 1:1000 in 1×
TBS, 5% milk, and 0.05% Tween-20. To visualize protein bands, the
ECL method was used; the protein level measurements were performed
by means of the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules,
CA).
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