14-3-3
https://medlineplus.gov/genetics/gene/ywhae/
Ywhae gene = 14-3-3 epsilon = tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (Ywhae)
Mus musculus tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (Ywhae), mRNA NCBI Reference Sequence: NM_009536.4
Dr. Yi Zhou's 14-3-3 FKO (Thy1-YFP-DIfopein) Transgenic Mice
See SFN abstracts (2010) 53.1/M17 and (2011) 790.09/FF16
From Kourtney Graham's Honor's Thesis:
In order to study the role of 14-3-3 proteins in the nervous system_ in vivo_, we have successfully created lines of neuronal-specific transgenic mice that may inhibit 14-3-3 dependent cellular processes. These transgenic mice express a YFP-fused peptide (R18) which binds to endogenous 14-3-3, and thus blocking its capability to interact with its binding partners; and are therefore considered to be a functional knockout. The transgenes are expressed under the control of the Thy-1 promoter which is neuronal-specific and produces variation of transgene expression in different brain regions, which is great among founder lines but minimal among descendants of a single founder (Feng et al. 2000). To minimize strain-dependent genetic variation, we have backcrossed the founder lines with wild-type C57BL/6 mice for at least 8 generations.
From Sun et al PMID 23861400 :
Glutamatergic kainate receptors (KAR) KAR-EPSCs at mossy fiber-CA3 synapses decay significantly faster in the 14-3-3 functional knock-out mice (but no difference in NMDA-R or AMPA-R EPSC in same synapses)
R18 peptide
R18 is a synthetic 21 amino acid unphosphorylated peptide (MW 2309.6) which binds to the same amphipathic groove on the surface of 14-3-3 as phosphorylated peptides, with high affinity for all 7 mammalian isoforms of 14-3-3 PMID 10493820. Commercial source of peptide Enzo #BML-P214-0001.
Difopein is a construct with two R18 monomers in a single 62 amino acid peptide PMID 11577088. Commercial source of peptide: Tocris #1245
R18 Sequence:
Pro-His-Cys-Val-Pro-Arg-Asp-Leu-Ser-Trp-Leu-Asp-Leu-Glu-Ala- Asn-Met-Cys-Leu-Pro-OH PHCVPRDLSWLDLEANMCLP
Difopein sequence (with individual R18 monomers in parentheses):
SADGA (PHCVP RDLSW LDLEA NMCLP) GAAGL DSADG A(PHCV PRDLS WLDLE ANMCL P) GAAG LE
C54 (consisting of the carboyl-terminal 54-amino acid region of theHMG-box binding protein Ste11p ) is another 14-3-3 inhibitor that is structurally unrelated to R18/difopein. The C54 region permits sequestration of Ste11p in the cytoplasm to inhibit nuclear localization PMID 12697825.
Thy-1 Promoter
YFP
YFP is 239 amino acids long (717 bp) according to plasmid map of Invitrogen Vivid ColorsTM pcDNATM/6.2 N-terminal EmGFP/YFP-DEST vectors see p, 28 of the manual. Difopein is 62 amino acids long (186 bp), so total YFP-difopein fusion protein should be ~ 301 amino acids (903 bp).
14-3-3 FKO papers
Vogt, Peggy, "Exploring the Function of 14-3-3 Proteins in Neurodegenerative Disease" (2011). Honors Theses. Paper 42.
C. Sun, H. Qiao, Q. Zhou, Y. Wang, Y. Wu, Y. Zhou, et al., Modulation of GluK2a Subunit-containing Kainate Receptors by 14-3-3 Proteins, J Biol Chem. 288 (2013) 24676–24690. PMID 23861400
Qiao H, Foote M, Graham K, Wu Y, Zhou Y. 14-3-3 proteins are required for hippocampal long-term potentiation and associative learning and memory. J Neurosci. 34(2014):4801-8. PMID 24695700
Antisera for IP Experiments
anti-pan-14-3-3 (K-19) , rabbit polyclonal, Santa Cruz #sc-629, 200 ug/1 ml, $279 datasheet
anti-HDAC4 (H-92), rabbit polyclonal, Santa Cruz #sc-11418, 200 ug/1 ml, $279 datasheet
anti-Torc1 (C71D11), rabbit mAb, Cell Signaling #2587 $225 datasheet