Goat anti rat igg

Flies were synchronized for 3 days under LD (500 lux:0 lux) cycles, and collected every 3–4 h. Protein was extracted from 40–50 male heads at each time point. Frozen heads were homogenized in 150 ml ice-cold extraction buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 5% glycerol, 10 mM EDTA, 0.1% Triton, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) plus protease inhibitors (complete mini Roche), phosphatase inhibitors cocktails 1 (0.5%) and 2-mM b-glycerophosphate (1%; Sigma). For SDS–PAGE, 50 ug of protein extracts were separated on 8–10% Tris–acetate gels (Invitrogen) and transferred to NC membranes. Primary antibodies used were mouse anti-ACTIN (abcam;1∶2000) and rat ani-CRY and rat anti-TIM (from the laboratory of Dr. Rosbash Lab; anti-CRY 1∶500; anti-TIM 1∶1000). HRP-conjugated secondary antibodies (Zhongshan Goldenbridge Biotechnology Co., Ltd) were goat anti-mouse IgG (1∶1000) and goat anti rat IgG (1∶1000). All western blots were performed in triplicate. Band intensity was calculated and analyzed with the Gel-Pro Analyzer 4.0.

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).