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