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. 2014 Apr 7;5:3628. doi: 10.1038/ncomms4628

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Copyright © 2014, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

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(a) Ca_V2.2 (top) co-immunoprecipitates with HA-tagged FMRP (bottom) in co-transfected tsA-201 cells, but not in control (Ab omitted). Ca_Vβ1b and Ca_Vα₂δ-1 were also transfected but neither were co-immunoprecipitated with HA-FMRP. Immunoblots were performed using Ca_V2.2, Ca_Vα₂δ-1, Ca_Vβ1b and HA Ab. Representative of three experiments. (b) Confocal images of in situ PLA showing interaction between transfected GFP-tagged Ca_V2.2 and HA-FMRP in tsA-201 cells. Ca_Vβ1b and Ca_Vα₂δ-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-Ca_V2.2 (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 Ca_V2.2 expressed in tsA-201 cells plus Ca_Vβ1b/Ca_Vα₂δ-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 Ca_Vα₂δ-1 nor Ca_Vβ1b expressed in tsA-201 cells (together with Ca_V2.2) were pulled down with FCT. Input represents 5% of protein input included in the assay. Immunoblots were performed using Ca_Vα₂δ-1 and Ca_Vβ1b Ab. Representative of three experiments. (f) Ca_V2.2 and mutant Ca_V2.2 (W391A) expressed in tsA-201 cells (with Ca_Vα₂δ-1/Ca_Vβ1b) 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 Ca_V2.2. (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.