Zebrafish Protein Expression Analysis

Western blot analysis was performed as previously described [36 (link),37 (link),38 (link)]. Briefly, zebrafish larvae were homogenized in ice-cold RIPA buffer to extract proteins. Each set of larvae (20 per experimental group of each experiment) was pooled for protein preparation such that n = 1 refers to protein from these 20 larvae. Protein concentrations were determined by the BCA method [39 (link),40 (link)]. After electrophoresis, the proteins were transferred from gels onto a polyvinylidene fluoride (PVDF) membrane 0.42 µm (GE Amersham, Casoria NA). Primary antibodies were incubated at 4 °C overnight, and antibody against Bax (Abcam ab32503, 1:800), bcl-2 (Abcam ab18285, 1:800), iNOS (Antibodies A323357, 1:500), and the proteins expression were normalized according to the expression of GAPDH (Abcam ab181602, 1:1000). After being washed three times with TBST, the membrane was incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit lgG or goat anti-mouse lgG (diluted at 1:5000) for 2 h at room temperature. Finally, the immunoreactive bands were detected using the ECL methods, and the protein bands were quantified by densitometry with BIORAD ChemiDocTM XRS+ software (Bio Rad, Hercules, CA, USA). The protein expressions were obtained by analyzing the density ratio of target proteins to GAPDH expression.