(a) CaV2.2 (top) co-immunoprecipitates with
HA-tagged FMRP (bottom)
in co-transfected tsA-201 cells, but not in control (Ab omitted).
CaVβ1b and CaVα2δ-1 were
also transfected but neither were co-immunoprecipitated with
HA-FMRP. Immunoblots
were performed using CaV2.2, CaVα2δ-1,
CaVβ1b and HA Ab. Representative of
three experiments. (b) Confocal images of in situ PLA showing
interaction between transfected GFP-tagged CaV2.2 and
HA-FMRP in tsA-201
cells. CaVβ1b and CaVα2δ-1 were
also transfected. PLA was performed using mouse FMRP Ab (7G1-1) and either rabbit
IgG (top row) or rabbit anti-GFP Ab (bottom row). PLA signals (white, right
panel) are detected only in cells positive for GFP-CaV2.2 (green, left
panel) when both GFP and FMRP Abs were used. 4',6-diamidino-2-phenylindole (blue) labels cell nuclei.
Scale bar, 10 μm. Representative of three experiments.
(c) Schematic depiction of FMRP and GST-fusion fragments used for pull-down assay.
Nter, N-terminus; Cter, C-terminus; KH1 and KH2, K-homology domains 1 and 2;
RGG, arginine-glycine-glycine box; FNT, GST-FMRP N-terminus; FCT,
GST-FMRP C-terminus;
aa, amino acid. (d) Western blots of pull-down assays show FCT, but
not GST alone or FNT, binds CaV2.2 expressed in tsA-201 cells plus
CaVβ1b/CaVα2δ-1.
Input represents 5% of protein input included in the assay. Western blots
(lower panel) show amount of GST-tagged protein used in the assay.
Representative of three experiments. (e) Western blots of pull-down
assays show neither CaVα2δ-1
nor CaVβ1b expressed in tsA-201 cells
(together with CaV2.2) were pulled down with FCT. Input
represents 5% of protein input included in the assay. Immunoblots were
performed using CaVα2δ-1
and CaVβ1b Ab. Representative of three
experiments. (f) CaV2.2 and mutant CaV2.2 (W391A) expressed
in tsA-201 cells (with CaVα2δ-1/CaVβ1b) were
both pulled down with FCT. Input represents 5% of protein input included in
the assay. Representative of three experiments. (g,h) Western
blots of pull-down assays show phosphorylation state of FMRP serine 499 does not modify
interaction with CaV2.2. (g) S499A,
dephosphomimetic-FCT; (h) S499D, phosphomimetic-FCT. Input represents
5% of protein input included in the assay. Representative of three
experiments. Full-size blots can be found in Supplementary Fig. 8.