Anti-DYKDDDDK polyclonal antibody

Mammalian zygote-mediated genome editing via the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system is widely used to generate genome-modified animals. This system allows for the production of loss-of-function mutations in various Y chromosome genes, including Sry, in mice. Here, we report the establishment of a CRISPR-Cas9-mediated knock-in line of Flag-tag sequences into the Sry locus at the C-terminal coding end of the Y chromosome (YSry-flags) In the F1 and successive generations, all male pups carrying the YSry-flag chromosome had normal testis differentiation and proper spermatogenesis at maturity, enabling complete fertility and the production of viable offspring. To our knowledge, this study is the first to produce a stable Sry knock-in line at the C-terminal region, highlighting a novel approach for examining the significance of amino acid changes at the naive Sry locus in mammals.

An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCF beta TrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein beta TrCP2, the substrate recognition subunit of SCF beta TrCP, was used as bait. beta TrCP2 wild type and the two mutants beta TrCP2-R447A and beta TrCP2-Delta F were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCF beta TrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T. Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCF beta TrCP ubiquitin ligase. Sci. Signal. 7 (2014) rs8-rs8,10.1126/scisignal.2005882. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license.