Primary antibody dilutions were prepared at 1:1000 to 1:500 in 5% BSA or dry milk in TBS with 0.05% v/v Tween. Protein samples prepared directly from lysate were prepped in 5X SDS sample buffer. Westerns were run in BioRad equipment in SDS buffer, using BioRad gradient gels, at 175 V for 42 min. Precision plus dual color protein marker (BioRad) was used in parallel to samples. Proteins were wet-transferred onto nitrocellulose membranes (0.2 µM, BioRad) at 100 V for 1 h. Membranes were blocked in 5% BSA or milk for 1 h RT depending on antibodies to be used. Membranes were incubated with primary antibodies overnight at 4° and washed for a total of 30 min in TBST before adding secondary diluted to 1:10,000 through 1:5,000 in 5% milk. Antibodies used in immunoblots are as follow: Anti-USP15 (Proteintech 14354-1-AP) used at 1:500-1:1000, Anti-Vinculin E1E9V XP (Cell Signaling Technologies #13901) used at 1:1000, Anti-KEAP1 P586 (Cell Signaling Technologies #4678) used at 1:500, Anti-NRF2 (Cell Signaling Technologies #12721) used at 1:500, anti-GAPDH D16H11 XP (Cell Signaling Technologies #5174) used at 1:1000, anti-pan-Actin (Cell Signaling Technologies #4968) used at 1:1000.
Anti usp15
Manufactured by Proteintech
Anti-USP15 is a laboratory antibody product designed for research purposes. It is an antibody that specifically targets and binds to the USP15 protein, which is a deubiquitinating enzyme involved in various cellular processes. The core function of this product is to allow researchers to detect and study the USP15 protein in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ubiquitin antibody specifically, by Merck Group (2 mentions)
Anti usp15 2d5, by Proteintech (2 mentions)
Anti mdm2, by Merck Group (2 mentions)
Precision plus dual color protein marker, by Bio-Rad (1 mentions)
Anti vinculin e1e9v xp, by Cell Signaling Technology (1 mentions)
Anti gapdh d16h11 xp, by Cell Signaling Technology (1 mentions)
Anti pan actin, by Cell Signaling Technology (1 mentions)
Anti nrf2, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Anti ubiquitin, by Cell Signaling Technology (1 mentions)
Anti ttf1, by Abcam (1 mentions)
Plx4032, by Selleck Chemicals (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Anti gst, by Cell Signaling Technology (1 mentions)
Anti gr1, by BioLegend (1 mentions)
Sr11302, by Selleck Chemicals (1 mentions)
Anti tbx3, by Abcam (1 mentions)
Anti tpo, by Abcam (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
5 protocols using anti usp15
1
Western Blot Analysis of Cellular Signaling Proteins
2
Investigating Ubiquitination and Deubiquitination of MDM2 and NFATc2
3
Investigating Ubiquitination and Deubiquitination of MDM2 and NFATc2
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Total cell lysates or subcellular extracts were prepared and subjected to IB assays as described41 (link). The Santa Cruz anti-USP15 (2D5) and the Proteintech anti-USP15 antibodies were used for detecting human and mouse USP15, respectively. The Millipore anti-MDM2 and the Sigma anti-MDM2 antibodies were used for detecting human and mouse MDM2, respectively. For ubiquitination assays, MDM2 and NF-ATc2 were isolated by IP under denaturing conditions39 (link), and the ubiquitinated MDM2 and NFATc2 were detected by IB using a ubiquitin antibody specifically detecting K48-linked ubiquitin chains (Millipore). For transfection models, MDM2 or NFATc2 was transfected into HEK293 cells along with HA-ubiquitin, and the ubiquitinated MDM2 and NFATc2 were isolated by denaturing IP and detected by anti-HA IB. For deubiquitination assays, the isolated MDM2–ubiquitin conjugates were incubated with purified USP15 or its catalytic mutant in a deubiquitination buffer42 (link) for 4 h and then subjected to IB using anti-ubiquitin.
4
Antibody Usage for Immunoblotting, IP, IHC, and IF
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Antibodies used for immunoblot (IB), immunoprecipitation (IP), immunohistochemistry (IHC) and immunofluorescence (IF) were as follows. Anti-TBX3 antibodies for IB Anti-TBX3 (#ab99302, Abcam, 1:500). Anti-Flag (#3165, 1:1000), Anti-α-Tubulin (T5168, 1:15000) was purchased from Sigma. Anti-HA (#3724, 1:1500), Anti-Ubiquitin (#3936, 1:500), Anti-GST (#2622, 1:1000), was purchased from Cell Signaling Technology. Anti-USP15 (#A300-923A, BETHYL, 1:1000), Anti-USP15 (#67557-1-Ig, Proteintech 1:200), Anti-Ki-67 (#ab15580, Abcam, 1:100), Anti-Tpo (#ab278525, Abcam, 1:200), Anti-Tg (#ab156008, Abcam, 1:200), Anti-Nis (#MA5-12308, Thermo, 1:400), Anti-Gr-1 (#108401, BioLegend, 1:200), Anti-PAX8 (#ab53490, Abcam, 1:10), Anti-TTF1 (#ab76013, Abcam, 1:200) were employed according to the instructions. Reagents used in this study included MG132 (#S2619, Selleck), Puromycin (#P8230, Solario), CHX (#HY-12320, MCE), Blasticidin (#B9300, Solario), PLX4032 (#S1267, Selleck), SCH772984 (#S7101, Selleck), SR11302 (#E2813, Selleck).
5
Immunohistochemical Analysis of USP15 Expression
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Two-step immunohistochemistry was used in our study. Firstly, tissue sections were treated to retrieval antigen using EDTA buffer; and then tissues were incubated with primary antibody anti- USP15 (1:800, Proteintech) at 4°C overnight. Next sections were incubated with secondary antibody (HRP-labeled anti-mouse antibody, DAKO), then visualized using diaminobenzidine (DAB) and hematoxylin re-dying after washed with PBS. The immunohistochemical results were valuated by pathologists, and scored as follows: negative for 0, “+” for 1, “++” for 2 and “+++” for 3. The positive staining rate was defined according to the proportion of positive stained cancer cells: “Negative” is 0, “1%-20%” for 1, “21%-40%” for 2, “41-60%” for 3, “61-80%” for 4, “81-100%” for 5. Take the product of “dyeing intensity” score and “dyeing positive rate” score as the total score.
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