Total protein lysates derived from transfected HEK293T cells were used for WB experiments using the human specific antibodies anti-TNKS1/2 (1:1000 dilutions; Santa Cruz Biotechnology; sc-8337); anti-USP25 (1:1000 dilution; Abcam, ab187156); anti-Flag (1:1000 dilutions; Sigma-Aldrich, F7425); and anti-Tubulin (1:5000 dilutions; Sigma, T5168). Membranes were developed with chemiluminescence substrate Pierce® ELC Western Blotting Substrate (Thermo Fisher Scientific, South Logan, UT, USA), and visualized on a LAS4000 device (Fujifilm, Tokyo, Japan). Protein quantification was done with the Image Gauge software (Fujifilm).
Anti usp25
Manufactured by Abcam
Sourced in United States
Anti-USP25 is a primary antibody that recognizes the Ubiquitin-specific protease 25 (USP25) protein. USP25 is a deubiquitinating enzyme involved in the regulation of various cellular processes. This antibody can be used to detect the expression of USP25 in samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti tnks1 2, by Santa Cruz Biotechnology (3 mentions)
Anti tubulin, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti gapdh, by Thermo Fisher Scientific (2 mentions)
Anti ha, by Roche (2 mentions)
β actin, by Abcam (2 mentions)
Anti usp28, by Abcam (2 mentions)
Pierce elc western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Image gauge software, by Fujifilm (1 mentions)
Las4000 device, by Fujifilm (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Anti hemagglutinin ha, by Merck Group (1 mentions)
Anti app, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti ubiquitin, by Santa Cruz Biotechnology (1 mentions)
Anti gfap, by Cell Signaling Technology (1 mentions)
Anti βiii tubulin, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Nitrocellulose filter membrane, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
7 protocols using anti usp25
1
Western Blot Analysis of Transfected HEK293T Cells
2
Immunoblot Analysis of Protein Targets
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Immunoblot analysis was performed as previously described (44 (link)). Antibodies were used as follows: anti-USP25 (Abcam, Cambridge, MA, USA; ab187156, 1:1000), anti-APP (Millipore, Billerica, MA, USA; MAB348, 1:1000), anti-Iba1 (Wako Pure Chemical, Osaka, Japan; 016-20001, 1:500), anti-GFAP (glial fibrillary acidic protein) (Cell Signaling Technology, Danvers, MA, USA; 3670, 1:1000), anti–β-III-tubulin (Abcam, ab18207, 1:1000), anti-HA (hemagglutinin) (Sigma-Aldrich, St. Louis, MO, USA; H6908, 1:1000), anti-ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-8017, 1:500), anti–β-actin (Xmbcss, Xiamen, China; bc001, 1:2000), and horseradish peroxidase (HRP)–conjugated secondary antibodies (Thermo Fisher Scientific, 31430 or 31460; 1:3000).
3
Western Blot for Cell Proteins
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Cell proteins were extracted and separated in SDS-PAGE gels, and transferred to nitrocellulose filter membranes (Millipore, USA). Western blot analyses were performed according to standard procedures. Western blot loading control was β-actin (Sigma-Aldrich, 1:30000). The following antibodies were used: anti-USP25 (Abcam, 1:1000), anti-E-cadherin (CST, 1:1000), anti-N-cadherin (CST, 1:1000).
4
Western Blot Analysis of Wnt Pathway Proteins
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HEK293T cells were cultured in DMEM (Gibco) with 10% (v/v) FBS (Gibco) and 1% penicillin/streptomycin. DLD-1, SW480, and HCT116 cells were maintained in RPMI1640 supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin. Cells were grown in a 37°C humidified incubator containing 5% CO2.
The commercial antibodies used for Western blotting analysis included the following: anti-USP25 (1:1000 dilution; Abcam, ab187156), anti-TNKS1/2 (1:1000 dilution; Santa Cruz Biotechnology, sc-8337), anti-Axin1 (1:1000 dilution; Cell Signaling Technology, no. 2087), anti-β-catenin (1:1000 dilution; Cell Signaling Technology, no. 9562), anti-Axin2 (1:1000 dilution; Cell Signaling Technology, no. 2151), anti-Flag (1:1000 dilution; Cell Signaling Technology, no. 2368), anti-Myc (1:1000 dilution; Proteintech, 16286-1-AP), anti-HA (1:1000 dilution; Proteintech, 51064-2-AP), and anti-Tubulin (1:10000 dilution; MBL, PM054). anti-Flag affinity gel (B23101) and anti-HA affinity gel (B23301) were from Biotool, CHX (no. 01810) was from Sigma, and XAV-939 (no. S1180) and MG132 (S2619) were from Selleckchem.
The commercial antibodies used for Western blotting analysis included the following: anti-USP25 (1:1000 dilution; Abcam, ab187156), anti-TNKS1/2 (1:1000 dilution; Santa Cruz Biotechnology, sc-8337), anti-Axin1 (1:1000 dilution; Cell Signaling Technology, no. 2087), anti-β-catenin (1:1000 dilution; Cell Signaling Technology, no. 9562), anti-Axin2 (1:1000 dilution; Cell Signaling Technology, no. 2151), anti-Flag (1:1000 dilution; Cell Signaling Technology, no. 2368), anti-Myc (1:1000 dilution; Proteintech, 16286-1-AP), anti-HA (1:1000 dilution; Proteintech, 51064-2-AP), and anti-Tubulin (1:10000 dilution; MBL, PM054). anti-Flag affinity gel (B23101) and anti-HA affinity gel (B23301) were from Biotool, CHX (no. 01810) was from Sigma, and XAV-939 (no. S1180) and MG132 (S2619) were from Selleckchem.
5
Deubiquitinase Profiling in NCI-H520 Cells
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Crude NCI-H520 cell extracts were prepared as described previously using glass-bead lysis in 50 mM Tris pH 7.4, 5 mM MgCl2, 0.5 mM EDTA, 250 mM sucrose, 1 mM DTT. For experiments with crude cell extracts, 50 μg of NCI-H520 cell lysate was incubated with different concentrations of USP28 inhibitor compounds (FT206 and AZ1) for 1 hr at 37°C, followed by addition of 1 μg HA-UbPA and incubation for 10 min (Figure 4B and C ) or 30 min (Figure 4—figure supplement 1A comparing FT206 and AZ1) at 37°C. Incubation with Ub probe was optimized to minimize replacement of non-covalent inhibitor FT206 by the covalent probe. Samples were then subsequently boiled in reducing SDS-sample buffer, separated by SDS-PAGE and analysed by Western blotting using anti-HA (Roche, 1:2000), anti-USP28 (Abcam, 1:1000), anti-USP25 (Abcam, 1:1000), anti-GAPDH (Invitrogen, 1:1000), or beta Actin (Abcam, 1:2000) antibodies. For cell-based DUB profiling, 5 × 106 intact cells were incubated with different concentrations of inhibitors in cultured medium for 4 hr at 37°C, followed by glass-bead lysis, labelling with HA-UbPA probe, separation by SDS-PAGE and Western blotting as described above.
6
Ectopic Expression of Flag-USP25 in HEK293T Cells
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The HEK293T cell line (CRL-1573; ATCC) was used for ectopic expression of Flag-USP25 and its mutants. HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St Louis, MO, USA), supplemented with 10% (v v−1) fetal bovine serum, 2μM l -glutamine, and 100 U mL−1 penicillin/streptomycin. Cells were grown in a 37 °C humidified incubator containing 5% CO2. The commercial antibodies used for western blotting (WB) analysis included the following: anti-TNKS1/2 (1:1000 dilutions; Santa Cruz Biotechnology; sc-8337); anti-USP25 (1:1000 dilution; Abcam, ab187156); anti-Flag (1:1000 dilutions; Sigma-Aldrich, F7425); and anti-Tubulin (1:5000 dilutions; Sigma, T5168), anti-Flag M2 affinity gel (Sigma-Aldrich, no. A2220). CHX (Sigma-Aldrich, no. 01810) was added to the cell culture medium in a final concentration of 100 μg mL−1 and cells were collected at the indicated times (0, 3, and 6 h) for WB. Bortezomib (Jansen Pharmaceuticals) was added to the cell culture medium in a final concentration of 0.5 μM and cells were collected after 6 h for WB.
7
Profiling Deubiquitinase Activity in Crude Cell Extracts
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Crude NCI-H520 cell extracts were prepared as described previously using glassbead lysis in 50 mM Tris pH 7.4, 5 mM MgCl2, 0.5 mM EDTA, 250 mM sucrose, 1 mM DTT. For experiments with crude cell extracts, 50 μg of NCI-H520 cell lysate was incubated with different concentrations of USP28 inhibitor compounds (FT206 and AZ1) for one hour at 37 °C, followed by addition of 1 μg HA-UbPA and incubation for 10 minutes (Figure 4B, 4C) or 30 minutes (Figure S4A comparing FT206 and AZ1) at 37 °C. Incubation with Ub-probe was optimised to minimise replacement of noncovalent inhibitor FT206 by the covalent probe. Samples were then subsequently boiled in reducing SDS-sample buffer, separated by SDS-PAGE and analysed by Western Blotting using anti-HA (Roche, 1:2000), anti-USP28 (Abcam, 1:1000), anti-USP25 (Abcam, 1:1000), anti-GAPDH (Invitrogen, 1:1000) or beta Actin (Abcam, 1:2000) antibodies. For cell-based DUB profiling, 5x10 6 intact cells were incubated with different concentrations of inhibitors in cultured medium for 4 hours at 37 °C, followed by glass-bead lysis, labelling with HA-UbPA probe, separation by SDS-PAGE and Western blotting as described above.
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