Ecl detection kit

Mouse polyclonal antibodies were raised against recombinant dZIP13-2 protein fragment (MTEEKMAKEGYKDPADSKLLRSGSADEENPQPKCVEIANCLLRRHGGQLPEGETSESCGGACDIEDVGKVCFLREQEQKSKERKEQPKRSGFSRWDAARAQKEEERKESIKQLE). Briefly, the cDNA fragments encoding the cytosolic side of this protein (dZIP13-2) were synthesized and cloned into pTwin1 (NEB) vector. The recombinant protein was expressed in E. coli and purified by chitin beads (NEB), and injected into mice for antibody generation (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China). The antibody was affinity purified and pre-absorbed before use. Anti-Fer2LCH was as described before (Tang and Zhou, 2013b (link)). Anti-tubulin rat monoclonal antibody (ab6160), anti-GM130 rabbit polyclonal antibody (ab30637), and anti-PDI mouse monoclonal antibody (ab2792) were obtained from Abcam (Cambridge, MA, USA). Secondary antibodies include HRP-conjugated goat anti-mouse IgG, goat anti-rabbit IgG and goat anti-rat IgG (Zhongshan Goldenbridge Biotechnology, Beijing, China). For Western blot analysis, fly samples were homogenized in the buffer containing 1% Triton X-100 plus 10% proteinase inhibitor cocktail (Sigma), centrifuged, separated on 10% SDS-PAGE, and transferred to nitrocellulose membranes (Millipore, Watford, UK). Signals were developed with ECL detection kit (Vigorous Biotechnology, Beijing, China).

SH-SY5Y cells were used to test the effect of metals on mammalian TH phosphorylation at Ser40. Antibodies of anti-tyrosine hydroxylase (AB152) and anti-tyrosine hydroxylase, phosphoSer 40 (AB5935) were purchased from Sigma-Aldrich (Shanghai, China). The secondary antibody HRP-conjugated goat anti-rabbit IgG was purchased from Zhongshan Goldenbridge Biotechnology (Beijing, China). For Western blot analysis, cells were homogenized in the buffer containing 1% Triton X-100 plus 10% proteinase inhibitor cocktail (Sigma), centrifuged, separated on 10% SDS-PAGE, and transferred to nitrocellulose membranes (Millipore, Watford, UK). Signals were developed with the ECL detection kit (Vigorous Biotechnology, Beijing, China). The experiments were repeated three times.