Western blot analysis of gcHIF-1α and gcHIF-4α proteins in hypoxic grass carp liver. (A) gcHIF-1α and gcHIF-4α proteins were synthesized by in vitro transcription and translation (IVTT) in E. coli lysate (Roche) and used as positive controls to test the specificity of the rabbit anti-gcHIF-1α (AB-1) and anti-gcHIF-4α (AB-4) polyclonal antibodies. No cross reactivity was detected with either of these antibodies. (B) Liver extracts were prepared from grass carps exposed to hypoxia (DO 0.5 ± 0.3 mg/l) for 0, 4 and 24 h and total protein (30 μg each) analysed by Western blotting for gcHIF-1α and gcHIF-4α using polyclonal antibodies AB-1 and AB-4, respectively. Signals were normalized against the β-tubulin protein. A representative Western blot from three different fish (n = 3) is shown.