Magic™ Mouse Anti-FLAG tag monoclonal antibody, clone AD

Mutations of dual serine/threonine and tyrosine protein kinase (DSTYK) are linked to congenital anomalies of the kidney and urinary tract (CAKUT). However, the function of DSTYK in the normal kidney and its role in the pathogenesis of CAKUT remain largely unknown. In this study, we investigated the role of DSTYK in the kidney by analyzing the consequences of Dstyk ablation in mice. We found that DSTYK deficiency resulted in swelling and diffuse distribution of F-actin cytoskeleton in proximal convoluted tubules. However, the architecture of F-actin cytoskeleton in the descending thin limb of Henel's loop, the thick ascending limb of Henel's loop, distal convoluted tubules, connecting tubules, collecting ducts and glomeruli was not affected following DSTYK deletion. The appearance of the urogenital system and the gross kidney anatomy of Dstyk(-/-) mice were normal. Also, no significant difference in the renal corpuscle's diameter and the density of glomeruli was found between Dstyk(+/+) and Dstyk(-/-) mice. To identify DSTYK binding partners, we generated a Flag-DSTYK knockin HEK 293T cell line with Flag tag sequence inserted to the N-terminus of endogenous DSTYK gene using CRISPR/Cas9 system. A total list of 67 proteins were identified by Co-immunoprecipitation (Co-IP)/mass spectrometry (MS) that potentially interact with DSTYK, mostly enzymes (39.0%), nucleic acid binding proteins (36.6%) and cytoskeletal proteins (14.6%). Profilin 1 and Profilin 2 were validated as bona fide DSTYK binding partners by Co-IP assay. These results uncover a role of DSTYK in the regulation of F-actin cytoskeleton in the mouse renal proximal convoluted tubules.

Streptomyces are of great biological and industrial significance due to their complex morphological development and ability to produce numerous secondary metabolites. However, the intrinsic biochemical mechanisms underlying morphogenesis and secondary metabolism are rarely revealed, partially because of the limited availability of the biochemical tools in Streptomyces. Here we provided series of integrative vectors with various affinity tags, including single tags 3 x FLAG, 3 x HA, 3 x Streptag II, 18 x His, 13 x Myc, and dual tags, all of which were driven from a strong constitutive promoter ermEp*. Using a sigma factor SigT from S. coelicolor as a model, we successfully expressed and immuno-detected SigT fused with all tags. Moreover, after SigT was N-terminally tagged with 3 x FLAG and C-terminally tagged with 18 x His, we isolated SigT-interactive proteins from the S. coelicolor lysate based on the tandem affinity purification (TAP). Particularly, among the proteins purified, the SigT cognate anti-sigma factor RstA ranked the top with the most total independent spectra. These data suggested the feasibility of these affinity tags in Streptomyces, which will be widely employed to explore the biochemical mechanisms to further understand the dynamic and elaborate regulation in this genus.