For Chromatin immunoprecipitation (ChIP) assay, SimpleChIP® Enzymatic Chromatin IP Kit was bought from Cell Signaling Technology (CST, Danvers, MA, USA). The crosslinked chromatin was digested with enzyme to break into 200–1000-bp fragments. Immunoprecipitation was performed using 2 μg of anti-XIAP (Abcam), 2 μg of anti-FUS (Abcam) and 2 μg of IgG with rotation all night at 4 °C. Then, 30 μl of magnetic beads were added into the reaction for 2 h at 4 °C. Immunoprecipitated chromatin was recovered and measured by qRT-PCR.
Anti fus
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-FUS is a primary antibody that binds to the FUS (Fused in Sarcoma) protein. FUS is an RNA-binding protein involved in various cellular processes, including transcription, splicing, and transport of mRNA. This antibody can be used in applications such as Western blotting and immunohistochemistry to detect and study the FUS protein.
Automatically generated - may contain errors
Lab products found in correlation
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Anti xiap, by Abcam (1 mentions)
Magnetic bead, by Merck Group (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti neun, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti eif2alpha, by Cell Signaling Technology (1 mentions)
Anti pur alpha, by Abcam (1 mentions)
Anti phospho eif2 alpha, by Cell Signaling Technology (1 mentions)
Anti tiar, by BD (1 mentions)
Anti fus, by Santa Cruz Biotechnology (1 mentions)
Anti ha, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
Anti pkg1, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti p stat3, by Cell Signaling Technology (1 mentions)
8 protocols using anti fus
1
Chromatin Immunoprecipitation Assay
2
RNA-Protein Interactions by RIP-Seq
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Briefly, cells were firstly cross-linked and lysed. Nuclei was isolated and incubated with primary antibodies at 4 °C overnight with Magnetic Beads (Millipore, Germany). The beads were washed and re-suspended in RIPA with Proteinase K and incubated for 1 h. TRIzol was used for RNA extractions. Anti-FUS (Abcam, 1:200) was used for RIP. Total RNAs and IgG were assayed simultaneously.
3
Immunochemistry Antibody Characterization
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Sources of commercial antibodies were as follows: anti-FUS (Santa Cruz, Dallas, TX, USA; 4H11), anti-Pur-alpha (Abcam, Cambridge, UK; ab77734), anti-Pur-alpha (Abcam; ab79936), anti-HA (Santa Cruz; Y-11), anti-HA (Santa Cruz; F-7), anti-Flag (Sigma-Aldrich, St. Louis, MO, USA; M2 and M2 affinity gel), anti-Flag fluorescein isothiocyanate (FITC) conjugated (Sigma-Aldrich; M2), anti-Phospho-eIF2-alpha (Cell Signaling Technology, Beverly, MA, USA; D9G8), anti-eIF2-alpha (Cell Signaling Technology; D7D3), anti-cleaved caspase-3 (Cell Signaling Technology; Asp175), anti-NeuN (Merck Millipore, Billerica, MA, USA; A60), anti-TIAR (BD Biosciences, Erembodegem, Belgium), anti-GAPDH (Chemicon-Merck Millipore), anti-puromycin 3RH11 monoclonal antibody (Kerafast, Boston, MA, USA), anti-FUS (Abcam; ab84078), anti-Islet-1/2 (DSHB, Iowa City, IA, USA; 39.4D5). Mouse monoclonal antibody specific for ribosomal protein S19 were prepared in our laboratory.31 (link) FITC, Rhodamine, and aminomethylcoumarin-conjugated affinity-purified secondary anti'bodies, selected for absent cross-reaction, were from Jackson ImmunoResearch (West Grove, PA, USA).
4
Western Blot Analysis of Protein Expression
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Total protein isolated from cells and tissues using RIPA lysis buffer (ProMab Biotechnology, USA). Equal amounts of protein were separated on 10% SDS polyacrylamide gels and then electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking in 5% skim milk, the membranes were incubated overnight at 4 °C with primary antibodies including anti-EMX2OS (1:400; Abcam, Cambridge, UK), anti-TCF12 (1:400; Abcam), anti-FUS (1:400; Abcam), anti-PKG1 (1:500; Cell Signaling Technology, Boston, MA), anti-PKG2 (1:500; Cell Signaling Technology) and β-actin (1:600; Abcam). Next, the membranes were incubated with proper horseradish peroxidase (HRP)-conjugated IgG for 1 h at 37 °C. Protein bands were visualized using an enhanced chemiluminescence detection system (ECL; Beyotime, Shanghai, China).
5
Immunofluorescence Staining of Cell Markers
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The PBS-rinsed cells were fixated by 4% paraformaldehyde and then permeabilized using 0.25% Triton X-100 in PBS, followed by blocking with 1% BSA. After being washed three times by PBS, cell incubation with primary antibodies was processed at 4°C overnight. Subsequently, cells were incubated with Cy3-conjugated secondary antibody for 1 h and the nuclei stained by DAPI. Images were captured under an immunofluorescence microscope (Olympus), with the analysis conducted by a Nikon and spot image acquisition system. The primary antibodies applied here were anti-E-cadherin (#14472, 1:50; Cell Signaling Technology), anti-N-cadherin (#13116, 1:200; Cell Signaling Technology), anti-FUS (ab124923, 1:100; Abcam), and anti-p-STAT3 (#9145, 1:100; Cell Signaling Technology).
6
RNase-Assisted RNA Interactome Profiling
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RNase-assisted RNA chromatography with RNAse A/T1 was performed, using in vitro-transcribed pri-mir-30c-1 and total MCF7 cell extracts28 (link). RNA-bound proteins were separated using 4–12% NUPAGE bis–tris system (Invitrogen) and individual lanes were subjected to mass spectroscopy (BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews). Results were confirmed by western blot analysis with anti-SRSF3 (MBL), anti-PARP-1 (Santa Cruz), anti-DDX17 (Santa Cruz), anti-hnRNPA1 (Thermo Scientific) and anti-FUS (Abcam) specific antibodies, as indicated above (Western blot analysis section).
7
FUS Protein Expression in HEK293T Cells
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HEK293T cells were transfected with 2 μg of the FUS DNA construct or 2 μg of a DNA construct without a transgene via the calcium chloride method. Cells were collected after two days. The samples were sonicated in radioimmunoprecipitation assay (RIPA) buffer [1% nonidet-P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), phosphate buffered saline (PBS)] with ethylenediamine tetraacetic acid (EDTA) and protease inhibitors (Pierce, Rockford, IL) and centrifuged. The supernatant was collected and protein content was determined by Bio-Rad Protein Assay Dye. Samples were normalized for protein content and electrophoresed in a 4–12% gradient polyacrylamide gel containing SDS (Bio-Rad). The primary antibody was anti-FUS (Abcam, Cambridge, MA), the goat anti-mouse secondary antibody was from Santa Cruz (Dallas, TX), and ECL reagents were from Amersham (Buckinghamshire, UK).
8
Immunohistochemical Analysis of Neural Markers
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Abcam: anti‐FUS (Abcam Cat# 84078, RRID:AB_2105201, 1:1000), anti‐GFP (Abcam Cat# ab13970, RRID:AB_300798, 1:1000); Cell Signaling: anti‐NeuN (Cell Signaling Cat# 24307, RRID:AB_2651140, 1:400); Millipore: anti‐ChAT (Millipore RRID:AB_2079751, 1:1000), anti‐GFAP (Millipore Cat# AB5541, RRID:AB_177521, 1:500), anti‐Olig2 (Millipore, RRID: AB_9610, 1:1000); Sigma: anti‐MMP9 (Sigma‐Aldrich Cat# M9570, RRID:AB_1079397, 1:60); Wako: anti‐Iba1 (Wako Cat# 019–19,741, RRID:AB_839504, 1:1000).
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