Please enable JavaScript.
Coggle requires JavaScript to display documents.
14-3-3/HCN1 (Characterizing consensus motifs (Characterizing consensus…
14-3-3/HCN1
Characterizing consensus motifs
Validation of consensus motifs
in vitro
14-3-3 PRED :check:
ITC :check:
thermal shift :check:
Characterizing consensus motif mutants
S657A; T659A
increased expression--transient :check:
no change stable line :check:
delta804-814
increased expression--transient :check:
no change --stable line :check:
delta861-871
increased expression -- transient :check:
slight increase in half-life (do not trust) :question:
increased expression-stable line :check:
no increase in half-life (N=1)
triple mutant
increased expression -- transient :check:
increased expression- stable line
increased surface expression :check:
increased current :check:
mRNA :star: :fire: :star: :fire:
AHA turnover :fire:
Glycosylation :fire:
Characterizing single S-A mutants
Clone S-A mutants
Do S-A mutants mimic site mutants?
Forced 14-3-3 binding :check:
clone R18 sequence insertion into 14-3-3 sites
14-3-3 interaction
14-3-3 pulldown
14-3-3 co-IP
HCN1 phosphorylation
Phospho HCN1 antibody
Retina western
Retina IHC
Dopamine
14-3-3 binding
Phospho HCN1 level
HCN1 expression/surface expression
BV02 (14-3-3 inhibition)
PKA activation
PKC activation
Unbiased site mapping
Fig 2 pt 1: Mapping interaction domain
Pulldown N/CT truncation mutants N=2 :check:
Truncate through CNBD and C-linker
Pulldown with CT reporter :check:
100AA truncations :fire:
600-700
600-800
600-910 (binds)
high resolution mutations
Pulldown with NT reporter
reporter design and cloning
FIg 1: Evidence of interaction
Pulldown Brain :check:
Pulldown HEK293T cells :check:
Pulldown retina (N=2) :check:
co-IP retina :check:
Controls
R18 competitive block :red_cross:
non-binding 14-3-3 mutant (rotation student) :star:
dephosphorylated pulldown (confusing result)
pulldown from membrane fraction
Priority experiments
To do
HCN1 turnover
Reporter pulldown assay (truncation)
HCN1-R18 expression
Rotation/undergrads
HCN1 mRNA expression
Order probe
Mutant 14-3-3 control
Go over cloning strategy