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HCN1/14-3-3 - 2020 update - Coggle Diagram
HCN1/14-3-3 - 2020 update
Biochemical characterization
Figure 1: evidence of interaction
IHC
Retina :check:
Whole Brain?
co-IP
Retina :check:
Whole Brain (not publication quality)
Pulldown
Retina (not publication quality)
Whole Brain :check:
Figure 3: mapping interaction
Reporter pulldown assay
100AA truncation (repeat to match other figures) :star:
30AA truncation :check:
just distal C-terminus :check:
distal C-terminus mutants (Δ804-814; Δ861-871 & S810A; S867A) :check:
Pulldown with full-length HCN1 vs HCN1 mutant
Figure 2: predicted binding sites
14-3-3 PRED :check:
ITC :check:
AA647-657 :check:
AA804-814 :check:
AA861-871 :check:
Biological role of interaction
Stable lines (validating) :star:
WT
T651A; S653A
Δ804-814
Δ861-871
T651A; S653A; Δ804-814
T651A; S653A; Δ861-871
Δ804-814; Δ861-871
T651A; S653A; Δ804-814; Δ861-871
S810A; S867A
Figure 4: HCN1 mutant expression
WB HCN1 in all 9 cell lines
Surface Biotinylation in select cell lines
Immunofluorescence in select cell lines
Figure 5: 14-3-3 binding sites stabilize HCN1
SNAP-tag pulse-chase assay
HCN1 expression after proteasome inhibition
Completed work, unsure where to place
Cell lines :check:
mRuby3-HCN1_wt
mRuby3-HCN1_triple mutant
mRuby3-HCN1_T651A; S653A
mRuby3-HCN1- Δ804-814
mRuby3-HCN1_ Δ861-871
HCN1 mutant characterization
total WB :check:
mRuby3-HCN1_wt
mRuby3-HCN1_T651A; S653A
mRuby3-HCN1_ Δ804-814
mRuby3-HCN1_ Δ861-871
mRuby3-HCN1_triple mutant
Surface biotinylation :check:
mRuby3-HCN1_wt
mRuby3-HCN1_triple mutant
Ih recordings (eurofins) :check:
mRuby3-HCN1_wt
mRuby3-HCN1_triple mutant