Whole tissue sections from LC03 and LC07 (4‐μm‐thick formalin‐fixed, paraffin‐embedded) were stained with primary antibodies (EpCAM (#2929, CST, 1:500), CD163(#93498, CST, 1:200), Topoisomerase IIα (#12286, CST,1:400), TRIM29(17542‐1‐AP, Proteintech, 1:100), DKK1(Abcam, ab109416, 1:800) sequentially and paired with TSA 7‐colour kit (abs50015‐100T, Absinbio). The order of antibodies/fluorescent dyes was showed as following: anti‐ EpCAM/TSA 480, anti‐ CD163/TSA 780, anti‐TOPIIα/TSA 620, anti‐TRIM29/TSA 520, anti‐DKK1/TSA 570 and then by staining with DAPI (D1306; Thermo Fisher). Pictures were scanned with Aperio Versa 8 tissue imaging system (Leica). Images were analysed using Indica Halo software.
Trim29
Manufactured by Proteintech
Sourced in United States
TRIM29 is a protein that functions as a regulator of gene expression. It contains a RING finger domain, a B-box type zinc finger domain, and a coiled-coil domain, which are typical structural features of the TRIM protein family. TRIM29 has been implicated in various cellular processes, including cell proliferation, apoptosis, and transcriptional regulation.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Epcam, by Thermo Fisher Scientific (1 mentions)
Cd163, by Thermo Fisher Scientific (1 mentions)
Aperio versa 8 tissue imaging system, by Leica (1 mentions)
Flag bead, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Sting, by Proteintech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
P iκb, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Mouse monoclonal antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
P pkc, by Cell Signaling Technology (1 mentions)
Cyclin e, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Phlpp1, by Proteintech (1 mentions)
Tfap2c, by Abcam (1 mentions)
4 protocols using trim29
1
Multiparametric Immunofluorescence Analysis of FFPE Tissue
2
Immunoblot and Immunoprecipitation Protocols
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For immunoblot assays, NPC or HEK293T cells were subjected to lysis in ice-cold low-salt lysis buffer (LSB, 150 mM NaCl, 50 mM Hepes pH 7.5, 1.5 mM MgCl2,1 mM EDTA, 10% glycerol, 1% Triton X-100) supplemented with 5 mg/mL protease inhibitor cocktail (Roche, Basel, Switzerland). Aliquots of 20–25 μL extracts were subjected to SDS-PAGE. For IP assays, NPC or HEK293T cells were transfected with corresponding expression vectors for indicated times, then FLAG- or Myc-tagged proteins were pulled down using FLAG-beads (Sigma -Aldrich, St. Louis, MO, USA) or Myc-beads (Immuoway, Newark, DE, USA). The following antibodies were used which were directed to: FLAG (Sigma, A8592), β-actin (Sigma, A2228), HA (Roche Applied Science, clone 3F10), Myc (Roche Applied Science, 11814150001), STING (Proteintech, IL, USA, 19851-1-AP), TRIM29 (Proteintech, 17542-1-AP).
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted using Pierce lysis buffer (Pierce, Rockford, IL). Protein quantification was performed using the Bradford method. A 50 μg sample of protein was added to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bradford, MA). Incubation of primary antibodies was performed overnight at 4°C. Antibody dilution was as follows: TRIM29 (1:800, Proteintech), p-PKC, p-IκB, cyclin D1, cyclin E, Bcl-2 (1:1,000, Cell Signaling, Boston, MA), and mouse monoclonal antibody against β-actin (1:2,000, Santa Cruz Biotechnology, Santa Cruz, CA). This was followed by incubation with HRP-coupled anti-mouse or rabbit IgG antibody (1:1,000, Cell Signaling Technology, Danvers, MA) at 37°C for 2 hours. Target proteins on polyvinylidene difluoride membrane were visualized using a Pierce ECL kit and captured using a DNR BioImaging System (DNR, Jerusalem, Israel).
4
IHC Analysis of Subcutaneous Tumor Samples
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The collected subcutaneous tumors were fixed with 4% formalin, embedded in paraffin and then sectioned into 4-μm-slices. The standard protocol for IHC of the TMAs and subcutaneous tumors using a streptavidin-peroxidase (SP) Kit (Zhongshan biotech, Beijing, China), as described previously [25 (link)]. The slides were incubated with antibodies specific for TFAP2C (Abcam, cambridge, MA, USA), TRIM29 (Proteintech, Chicago, IL, USA), PHLPP1 (Proteintech, Chicago, IL, USA), p-AKT (Cell Signaling Technology, Danvers, MA, USA) and Ki-67 (Cell Signaling Technology, Danvers, MA, USA). The images of IHC staining were obtained by an Olympus microscope (Tokyo, Japan).
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