Fig. 6. Formation of FMRP granules involves FMRP dimerization.

(A–C) Co-immunoprecipitation of GFP-dFMRP mutant polypeptides with endogenous dFMRP. (A) Schneider cells were transfected with GFP-dFMRP or GFP-ΔPP. 48 h post transfection, cells were lysed and their extracts used to immunoprecipitate dFMRP using anti-GFP antibodies. IgG were used as a control immunoprecipitation. IP: immunoprecipitate; total represents 5% of the input used for immunoprecipitation. Immunoprecipitated proteins were analyzed by western blot for dFMRP using anti-dFMRP antibodies. The positions of GFP-dFMRP, GFP-ΔPP, and endogenous dFMRP are indicated by arrows. (B,C) Validation of GFP-dFMRP interaction with endogenous dFMRP. Schneider cells were first treated with siRNAs specific to the 3′UTR of dFMR1 mRNA and were then transfected with GFP-dFMRP 48 h later. Twenty-four h later, cells were lysed and their extracts used to immunoprecipitate dFMRP using anti-GFP antibodies. (B) Total: 5% of the input used for immunoprecipitation. Proteins were analyzed by western blot for dFMRP and GFP-dFMRP expression using anti-dFMRP antibodies Tubulin serves as a loading control. (C) IP: immunoprecipitate. Immunoprecipitated proteins were analyzed by western blot for dFMRP using anti-dFMRP antibodies. The positions of GFP-dFMRP and endogenous dFMRP are indicated by arrows. (D–G) GFP-I304N is a weak inducer of FMRP granules in STEK cells. MEF and STEK cells were transfected with either GFP-hFMRP or GFP-I304N (D,F) for 48 h. Cells were then processed for immunofluorescence to detect GFP fusion proteins (green). The intracellular localization of the FMRP partner FXR1 in FMRP granules (red) was revealed using specific antibodies. The indicated percentage of FMRP granules induced by expressing either GFP-hFMRP (D,E) or GFP-I304N (F,G) in MEFs and STEK is representative of 3 different experiments. Scale bars in D,F: 10 µm.