Cells used in this study were seeded on 24-well plates, and transfected the following day with EBOV RNP-expressing plasmids as described above, either in combination with 150ng of individual ISG-expressing plasmids per well or followed by the addition 4 hours post-transfection of increasing amounts of type-I interferon. 24 hours later cellular lysates were subjected to SDS-PAGE and western blots performed using mouse monoclonal antibodies anti-HSP90 (Santa Cruz), anti-HA (Covance) or anti-TRIM25 (Abcam), or rabbit antibodies anti-EBOV NP, anti-EBOV VP35, anti-EBOV VP30, anti-TRIM25 (Abcam), anti-RIG-I (Enzo), anti-MAVS/VISA (Bethyl), anti-TBK1 (Abcam), anti-ZCCHV/ZAP (Abcam) or anti-NPC1 (Thermo Fisher). Visualizations were done by Image-Quant using either HRP-linked anti-mouse or anti-rabbit secondary antibodies (Cell Signaling).
Anti trim25
Manufactured by Abcam
Sourced in United States
Anti-TRIM25 is a primary antibody that recognizes the TRIM25 protein. TRIM25 is a member of the tripartite motif (TRIM) family of proteins and functions as an E3 ubiquitin ligase. It plays a role in antiviral signaling pathways and innate immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (2 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Anti hsp90, by Santa Cruz Biotechnology (1 mentions)
Anti rig 1, by Enzo Life Sciences (1 mentions)
Anti tbk1, by Abcam (1 mentions)
Anti ha, by Fortrea (1 mentions)
Anti actin, by Abcam (1 mentions)
Anti tgf β, by Abcam (1 mentions)
Anti tubulin, by Cell Signaling Technology (1 mentions)
Anti smad4, by Cell Signaling Technology (1 mentions)
Anti smad2, by Cell Signaling Technology (1 mentions)
Anti p smad2, by Cell Signaling Technology (1 mentions)
Hyperfilm, by GE Healthcare (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
Gotaq polymerase, by Promega (1 mentions)
Modifying enzymes, by Thermo Fisher Scientific (1 mentions)
Sc 20559, by Santa Cruz Biotechnology (1 mentions)
Z vdvad fmk, by R&D Systems (1 mentions)
Sc 166926, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 2, by BD (1 mentions)
7 protocols using anti trim25
1
EBOV Regulation by Host Factors
2
Antibody Characterization for TGF-β Signaling
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The following antibodies were used in the present study: anti-TRIM25, anti-actin, anti-TGF-β and anti-bone morphogenetic protein (BMP)-4 and purchased from Abcam (Cambridge, MA, U.S.A.). Anti-p-Smad2, anti-Smad2, anti-p-Smad4, anti-Smad4, and anti-tubulin were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.).
3
Apoptosis Regulation by TRIM25 and Caspases
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Doxorubicin was purchased from Biotrend Chemicals (Cologne, Germany). Actinomycin D and rapamycin were obtained from Calbiochem (Schwalbach, Germany) and Merck KGaA (Darmstadt, Germany). The caspase-2-specific chemical inhibitor (Z-VDVAD-FMK) was from R & D Systems (Wiesbaden, Germany). Antibodies used in this study included the following: Anti-TRIM25 (#115737) from Abcam (Berlin, Germany) and (sc-166926) from Santa Cruz (Heidelberg, Germany), respectively. Anti-caspase-2 (#611022) from BD Biosciences (Heidelberg, Germany), anti-caspase-3 (#9662), anti-phospho-S6 ribosomal protein (Ser240/244, #2215), and anti-cytochrome c (#4272) from Cell Signaling (Frankfurt, Germany), anti-β-actin (#A2228) were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Secondary Horseradish Peroxidase HRP-linked antibodies sc-2054 and sc-20559 were obtained from Santa Cruz and a Cy5-coupled goat anti-mouse antibody was derived from Life Technologies (Darmstadt, Germany). Hyperfilm and the ECL system were purchased from GE Healthcare (Freiburg, Germany). Go-Taq polymerase was from Promega (Mannheim, Germany). Medium and supplements as well as modifying enzymes were from Invitrogen (Karlsruhe, Germany).
4
Immunohistochemical Analysis of TRIM25 and IGF2BP3
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IHC was performed as described previously [24 (link)]. Tissue sections (4 μm) were deparaffinized with xylene and hydrated using an alcohol gradient. Endogenous peroxidase-blocking and antigen retrieval were performed sequentially. The sections were incubated with polyclonal rabbit anti-TRIM25 (diluted 1:200, Abcam, MA, USA) and anti-IGF2BP3 (diluted 1:200; Abcam, MA, USA) followed by incubation with a secondary antibody IgG (ZSGB-BIO, China). Histological examination was performed using 3,3′-diaminobenzidine kit (DAB, OriGene, China) and hematoxylin. If the proportion of positive cells was >50% in 5 random fields, the specimen was considered to show high TRIM25/IGF2BP3 expression.
5
Comprehensive Western Blotting Protocol
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Western blotting was performed according to a standard protocol, as described previously63 (link). The following primary antibodies were used at a dilution of 1:1000: anti-OTUD5 (D8Y2U, Cell Signaling Technology), anti-TRIM25 (Abcam, ab167154), anti-PML (Abcam, ab53773), anti-HA (H9658, Sigma), anti-Flag (F3165, Sigma) and anti-ubiquitin (SC-8017, Santa Cruz). Anti-β-actin (Sigma, AC-15) primary antibody was used at a dilution of 1:5000. Goat anti-Rabbit IgG Secondary Antibody HRP conjugated (SAB Signalway Antibody, L3012) and Goat anti-Mouse IgG Secondary Antibody HRP conjugated (SAB Signalway Antibody, L3032) were used at a dilution of 1:5000.
6
Quantitative Protein Analysis of Muscle Lysates
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Equal amounts of protein from each muscle lysate (∼60 µg) were separated using the TGX stain-free sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) system from Bio-Rad (Hercules, CA, USA). Proteins were transferred onto a polyvinylidene difluoride membrane using the Bio-Rad TransBlot Turbo system and detected with the following commercial antibodies: rabbit polyclonal anti-Akt (Cell Signaling, #9272, 1:1000), rabbit monoclonal USP14 (Cell Signaling, #11931, 1:1000), mouse monoclonal anti-α-tubulin (Sigma Aldrich, T9026, 1:500) and rabbit monoclonal anti-TRIM25 (Abcam, ab167154, 1:000). An anti-dystrophin antibody (Dys1, Novocastra, NCL-DYS1) was used as a loading control. Proteins were visualized using a chemiluminescence system (Amersham ECL Prime) from GE Healthcare.
7
Western Blot Analysis of Cell Proteins
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Proteins resolved by SDS-PAGE were transferred to Immobilon-P PVDF membrane (Millipore, Burlington, VT, USA). Blots were probed with anti-DDX3X (GeneTex #GTX110614; 1:1000 v/v), anti-TRIM25 (Abcam #ab86365; 1:1000 v/v), anti-FLAG (Cell Signalling Technology, Danvers, USA, CST #8146S, #73916S; 1:1000 v/v, SCBT #sc-166355; 1/200 v/v), anti-HA (CST #2367S, #3724S; 1:1000 v/v), anti-mCherry (1:1000 v/v), anti-myc (CST #5605S; 1:1000 v/v), anti-ubiquitin (CST #3936; 1:1000 v/v) or anti-β-actin HRP-conjugate (CST #5125; 1:5000 v/v) antibodies, followed by HRP-conjugated anti-rabbit (Millipore, Burlington, USA #12-348; 1:10,000 v/v) or anti-mouse (CST #7076; 1:10,000 v/v) antibodies. The 5% (w/v) skim milk in TBS with 0.1% (v/v) Tween-20 was used to block and wash membranes and was used as the antibody diluent.
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