Amyloid beta (Aβ) 1–42 and scrambled Aβ peptide were from rPeptide. Dimethyl sulfoxide (DMSO) was from Fisher Scientific. Okadaic acid was from Sigma. The antibodies used in this study were purchased from Santa Cruz Biotechnology (anti-PSD95), GenScript Corporation (anti-Gapdh), Millipore (anti-puromycin), Abcam (anti-synapsin-I, anti-PSD95, anti-p-eIF4E and anti-MAP2) and Cell Signaling (anti-eIF2A, anti-p-eIF2A, anti-eIF4E, anti-PDI, anti-FMRP, anti-eEF2, anti-p-eEF2, anti-PP1, anti-PP2A-A, anti-PP2A-B, and anti-PP2A-C). HRP-conjugated secondary antibodies were from Cell Signaling and Jackson ImmunoResearch.
Anti fmrp
Manufactured by Cell Signaling Technology
Anti-FMRP is a primary antibody product that targets the Fragile X Mental Retardation Protein (FMRP). FMRP is an RNA-binding protein that plays a crucial role in regulating gene expression and neuronal function. The Anti-FMRP product can be used to detect and analyze the expression and localization of FMRP in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti neun, by Cell Signaling Technology (2 mentions)
Anti gfap, by Cell Signaling Technology (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Okadaic acid, by Merck Group (1 mentions)
Anti puromycin, by Abcam (1 mentions)
Anti psd95, by Cell Signaling Technology (1 mentions)
Anti p eif4e, by Cell Signaling Technology (1 mentions)
Anti map2, by Cell Signaling Technology (1 mentions)
Anti eif2a, by Cell Signaling Technology (1 mentions)
Anti p eif2a, by Cell Signaling Technology (1 mentions)
Anti eif4e, by Cell Signaling Technology (1 mentions)
Anti pdi, by Cell Signaling Technology (1 mentions)
Anti eef2, by Cell Signaling Technology (1 mentions)
Anti p eef2, by Cell Signaling Technology (1 mentions)
Anti pp2a a, by Cell Signaling Technology (1 mentions)
Anti pp2a b, by Cell Signaling Technology (1 mentions)
Anti pp2ac, by Cell Signaling Technology (1 mentions)
Anti synapsin 1, by Cell Signaling Technology (1 mentions)
Anti psd95, by GenScript (1 mentions)
6 protocols using anti fmrp
1
Aβ Peptide Preparation and Antibody Analysis
2
Co-immunoprecipitation of TDP-43 and FMRP
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Cells/forebrain tissue were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail tablet (Roche). Extracts were quantified with the Bradford protein assay (Bio-rad). Protein extracts (0.5 mg-1 mg) were diluted to 0.5 mg/ml with lysis buffer.
Co-immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore) according to the manufacturer's instructions with either 4 μg anti-TDP-43 (Proteintech, Cat# 10782-2 AP) or 4 μg of anti-FMRP (Cell Signaling Technology, Cat# 4317). For western blot analysis, precipitated proteins were washed and subsequently eluted in denaturation buffer and heated at 94 °C for 3 min. For RNA-immunoprecipitation analysis, precipitated proteins were washed and subsequently eluted in non-denaturation buffer, and RNAs in the immunoprecipitates were purified using Quick-RNA™ Microprep Kit (ZYMO RESEARCH) according to the manufacturer's instructions.
Co-immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore) according to the manufacturer's instructions with either 4 μg anti-TDP-43 (Proteintech, Cat# 10782-2 AP) or 4 μg of anti-FMRP (Cell Signaling Technology, Cat# 4317). For western blot analysis, precipitated proteins were washed and subsequently eluted in denaturation buffer and heated at 94 °C for 3 min. For RNA-immunoprecipitation analysis, precipitated proteins were washed and subsequently eluted in non-denaturation buffer, and RNAs in the immunoprecipitates were purified using Quick-RNA™ Microprep Kit (ZYMO RESEARCH) according to the manufacturer's instructions.
3
Investigating Synaptic Protein Regulation
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Antibodies used in this study are as follows: anti-GluA1, anti–hnRNP A2/B1, anti–PSD-95 (Abcam), anti–phospho-RPS6, anti–phospho-ERK (extracellular signal–regulated kinase), anti-FMRP (Cell Signaling), anti–N-term-GluA1, anti–N-term-GluA2, anti–hnRNP D (Millipore), anti-MAP2, anti–hnRNP A1, anti–14-3-3ζ (Santa Cruz), anti–hnRNP Q, anti-Flag (Sigma-Aldrich), anti-NMDAR1 (Synaptic Systems), and anti-actin (MPBIO). To inhibit translation, SHSY5Y cells were treated with 10 nM RAD001 or cycloheximide (100 μg/ml) and harvested at the indicated times. To block cap-dependent translation or induce synaptic stimulation, neurons were treated with 20 nM rapamycin (Sigma-Aldrich) or recombinant BDNF (100 ng/ml) (PeproTech), respectively.
4
Western Blot Analysis of Neural Markers
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Treated cells and brain tissues were lysed in the TNEN lysis buffer (25mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, and 0.1% SDS) supplemented with the protease inhibitor. Equal amounts of protein lysates were subjected to SDS-polyacrylamide gel electrophoresis and proteins were detected by the indicated antibodies. Primary antibodies used were as follows: anti-FMRP (Cell Signaling Technology, 4317S, 1:1,000), anti-NeuN (Cell Signaling Technology, 94403S, 1:1,000), anti-GFAP (Cell Signaling Technology, 3670S, 1:1,000), anti-Iba1 (Wako, 016–20001, 1:1,000), anti-GAPDH (Abways, AB0038, 1:5,000), and anti-α-tubulin (Millipore, MABT205, 1:10,000). HRP-conjugated secondary antibodies used were goat anti-rabbit IgG (H + L), HRP (Thermo Fisher Scientific, 31460, 1:5,000) and goat anti-mouse IgG (H + L), HRP (Thermo Fisher Scientific, 31430, 1:5,000).
5
Immunohistochemical Analysis of FMRP, NeuN, and GFAP in Mouse Brain
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Treated mice were anesthetized and intracardially perfused with ice-cold PBS. The brains were dissected quickly and post-fixed in 4% paraformaldehyde at 4°C for 24 h, and cryoprotected in 30% sucrose. Dehydrated tissues were frozen in OCT and cut into 15-μm-thick sections using a freezing microtome (Leica). Mouse coronal slices were blocked in 5% BSA and permeabilized in 0.2% Triton X-100 diluted in PBS at room temperature, and incubated with indicated primary antibodies: anti-FMRP (Cell Signaling Technology, 4317S, 1:50), anti-NeuN (Cell Signaling Technology, 94403S, 1:200), and anti-GFAP (Cell Signaling Technology, 3670S, 1:200) at 4°C overnight. Slices were then stained with fluorescence-conjugated secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A-11008, 1:400) or Alexa Fluor 594 goat anti-mouse IgG (H + L) (Thermo Fisher Scientific, A-11005, 1:400) for 1 h at room temperature in the dark. Confocal images were captured with the FV1000MPE-B (Olympus) confocal microscope.
6
Isolation and Analysis of Neuronal Proteins
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Protein extracts from cortical neurons or tissues were prepared with sample buffer [60 mM tris-HCl (pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol, and 0.1% bromophenol blue] and boiled for 5 min. For FACS sorting, the whole cortex dissected from WT or Fmr1 KO was homogenized by douncing followed by nuclei extraction via sucrose gradient ultracentrifugation (69 (link)). The nuclei were recovered from the pellet, resuspended, and incubated with NeuN antibody (Millipore, MAB377). Immunotagging with anti-NeuN conjugated to Alexa Fluor 488 (Invitrogen, A-21202) allows for sorting of the NeuN+ neuronal nuclei by fluorescence-activated sorting through a FACS machine (MoFlo Astrios, Beckman Coulter), followed by Western blotting. Proteins were separated by SDS-PAGE and subjected to Western analysis using the following antibodies: anti-BRD2 (Bethyl, A302-583A), anti-BRD3 (Active Motif, 61489) or anti-BRD4 (Bethyl, A301-985A100), anti-CBP (Santa Cruz, sc-369x), anti-FMRP (Cell Signaling, 4317S), anti–β-actin (Santa Cruz, sc-47778), anti–α-tubulin (Cell Signaling, 2144S), anti-H3K27ac (Abcam, Ab4729), anti-H4K8ac (Abcam, Ab15823), anti-H3K4me1 (Abcam, Ab8895), anti-H3ac (Millipore, 06-599), or anti-H3 (Abcam, ab176842).
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