Molecular Characterization of Two Mitogen-Activated Protein Kinases: p38 MAP Kinase and Ribosomal S6 Kinase From Bombyx mori (Lepidoptera: Bombycidae), and Insight Into Their Roles in Response to BmNPV Infection

Muhammad, Azharuddin; Toufeeq, Shahzad; Yu, Hai-Zhong; Wang, Jie; Zhang, Shang-Zhi; Li, Bing; Li, Zhen; Yang, Li-Ang; Hu, Pei; Ma, Yan; Xu, Jia-Ping

The recombinant Bmp38 and BmS6K proteins in purified form were submitted to HuaAn Biotechnology Ltd. (HUABIO, Hangzhou, China) , to raise rabbit antibody as per protocol described by Yu et al. (2017a) . Similarly, proteins for western blotting proteins were extracted from the various tissues of silkworms as per instructions of previous study (Yu et al. 2017a ). Using a BCA protein assay kit (Bio-Rad) the purified proteins concentrations were quantified. In short, 12% SDS-PAGE gel was used for the separation of prepared protein samples (60 µg). It was then transferred into membranes of polyvinylidenedifluoride (PVDF). PVDF membrane was blocked overnight using 5% nonfat milk in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM K 2 HPO 4 , pH 7.5, 0.1% ). It was washed three times with PBST, then used primary antibody (rabbit anti-Bmp38 and anti-BmS6K; diluted 1:5000), and incubated at room temperature for 2 h. Antigen-antibody complexes were detected after washing, with a horseradish peroxidase (HRP)conjugated goat anti-rabbit secondary antibody (1:5,000 dilution; HUABIO, Hangzhou, China) in blocking buffer for time period of 1 h. Immobilized conjugates were visualized on membranes in HRP substrate solution (Tiangen, Beijing, China). GAPDH monoclonal antibody was used as control (Transgene Biotech).