We quantified colocalization between FXR1P and ribosomal proteins using the Intensity Correlation Analysis Plugin in ImageJ [37]. adjusted equally on the images to demonstrate the level of background staining from the poly (dA) probe. Scale bars?=?10 m.(TIF) pone.0026120.s002.tif (944K) GUID:?9270049F-4D1E-4142-9CC6-6E8E75E87163 Figure S3: FXR1P partially colocalizes with FMRP, FXR2P and Argonaute 2 in clusters along the dendrite. Immunostaining of dissociated hippocampal neurons at 14 days with anti-FXR1P (#ML13) and A. anti-FMRP (1C3), B. anti-FXR2P (A42) and C. anti-Ago2 antibodies demonstrates partial colocalization of FXR1P with these three known interacting proteins (P0 staining is also shown for comparison). Note that Ago2 also DLL3 shows complementary staining with FXR1P, with Ago2 more likely to be found at the edges of the P0 clusters and FXR1P in the center. Graphs with labeled peaks demonstrating the covariance Anamorelin HCl (or complementary staining in the case of Ago 2) in the fluorescence intensities along the dendritic segment are shown at the right. Scale bars?=?10 m.(TIF) pone.0026120.s003.tif (2.1M) GUID:?E0329BDF-5873-49AC-BB08-8D0639ADE0E6 Figure S4: TIA-1 redistributes to stress granules. A. COS-7 cells were treated with 20 g/ml puromycin for 2 hours, followed by immunostaining for TIA-1. A small percentage of COS-7 cells display clearly visible TIA-1 positive cytoplasmic granules. Scale bar?=?10 m. B. Dissociated hippocampal neurons were treated with 0.5 mM arsenite Anamorelin HCl for 30 minutes and immunostaining for Anamorelin HCl TIA-1. Neurons showed the characteristic redistribution of TIA-1 into cytoplasmic granules. Scale bar?=?10 m.(TIF) pone.0026120.s004.tif (1.4M) GUID:?A0DF06FA-1C8A-4ADC-8402-33F4044C4D1E Figure S5: Fragile X Proteins colocalize with each other. Dissociated hippocampal neurons were transfected with A. eGFP-FXR1P, B. eGFP-FXR2P and C. eGFP-FMRP at 7 days and and instead of (discrepancy is underlined; see [28]). This discrepancy leads to an amino acid change of MAEL to MADV, which corresponds to the original reported sequence for human FXR1P (Accession number: “type”:”entrez-protein”,”attrs”:”text”:”AAC50155.1″,”term_id”:”887793″,”term_text”:”AAC50155.1″AAC50155.1, see [11]). This discrepancy was corrected in the pcDNA3.1-FXR1P-myc-his construct. Expression from all constructs was driven by the CMV promoter. Antibodies For detecting FXR1P, we used a rabbit polyclonal antibody against FXR1P (#ML13) which has been described previously [31]. Other antibodies used included mouse monoclonal antibodies against FMRP (mAb1C3;[32]), FXR1P (mAb3FX;[22]), FXR2P (mAbA42, Abcam), myc (Santa Cruz; 9E10), MAP-2 (Sigma-Aldrich; HM-2)and GAPDH (Abcam; ab9484), human anti-ribosomal P antibodies (Immunovision), a rabbit anti-ribosomal large protein L7 (Cell Signaling), a rabbit monoclonal antibody against S6 (Cell Signaling; 5G10) and a goat polyclonal antibody against TIA-1 Anamorelin HCl (Santa Cruz; sc-1751). The specificity of the anti-ribosomal P antibodies for the large ribosomal subunits P0, P1 and P2 was verified previously by others [24]. HEK cell culture, transfection and western blotting Human embryonic kidney cells with the SV-40 T antigen (293-T) were Anamorelin HCl cultured in high glucose Dulbeccos Modified Essential Medium (DMEM, Invitrogen) containing L-glutamine, 110 mg/L sodium pyruvate, 10% fetal bovine serum and 1% penicillin-streptomycin. One day before transfection, cells were split and plated at a density of 1 1.2106 cells per 6 cm dish. Cells were transfected with various Fragile X plasmids using Polyfect (Qiagen) according to the manufacturers instructions. Cells were lysed after 48 hours in 400 l RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mM Tris pH 8.0, 150 mM NaCl and 1 mM EDTA) containing 1 g/ml each of leupeptin, aprotinin, pepstatin, 10 mM NaF, 1 mM sodium ortho-vanadate and 1 mM PMSF. Lysates were diluted with 3X sample buffer and equal quantities of each lysate were run on a 10% polyacrylamide gel and transferred to PVDF membranes following standard protocols. Membranes were blocked for 40 minutes with 5% BSA/TBS-0.1% Tween, and incubated overnight at 4C with either #ML13 (1100,000) or anti-myc (12000) in TBS-0.1% Tween. The next day membranes were incubated for 1 hour at room temperature with secondary antibodies conjugated to HRP. Chemiluminescent signal was obtained using Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare) and captured on X-ray film. Hippocampal Lysates and.