Anti uchl1

Manufactured by R&D Systems

Anti-UCHL1 is a laboratory reagent used for the detection and quantification of UCHL1 (Ubiquitin Carboxyl-Terminal Hydrolase L1) in various biological samples. UCHL1 is an enzyme involved in the ubiquitin-proteasome pathway, which plays a role in protein degradation. This reagent can be used in techniques such as Western blotting, immunohistochemistry, and ELISA to analyze the expression and distribution of UCHL1.

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UCHL1 (5 citations)

3 protocols using anti uchl1

Immunofluorescence Assay for HIF-1α and UCHL1 in 2D Cell Culture

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For HIF‐1α and UCHL1 staining in 2D culture, cells treated with each condition were washed with PBS (−) and fixed in 4% paraformaldehyde at room temperature for 15 minutes, followed by permeabilization with 0.1% Triton X‐100 for 10 minutes on ice. The coverslips were blocked with 3% BSA for 12 hours and incubated with anti–HIF‐1α (1:500; BD Biosciences) and anti–UCHL1 (1:500; R&D Systems) antibodies for 1 hour at 4°C. After three 5‐minute washes with PBS (–), samples were probed with goat anti–rabbit polyclonal secondary antibody (Alexa Fluor 488, 1:1000; Abcam) or goat anti–mouse polyclonal secondary antibody (Alexa Fluor 568, 1:1000; Abcam) mixed with 4′,6‐diamino‐2‐phenylindole (DAPI) (1:5000, Roche) at room temperature for 1 hour. The coverslips were mounted onto slides with Dako Fluorescence Mounting Medium (Agilent). Fluorescence was detected by fluorescence microscopy (Olympus).

Li X., Hattori A., Takahashi S., Goto Y., Harada H, & Kakeya H. (2019). Ubiquitin carboxyl‐terminal hydrolase L1 promotes hypoxia‐inducible factor 1‐dependent tumor cell malignancy in spheroid models. Cancer Science, 111(1), 239-252.

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Investigating UCHL1 and Cell Cycle Regulation

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Anti-cyclin B1 from Santa Cruz (sc-245) was used for immunohistochemistry, western blot, immunoprecipitation, immunofluorescence and the proximity ligation assay. Anti-UCHL1 from Sigma-Aldrich (HPA005993) was used for immunohistochemistry, immunofluorescence and proximity ligation assay; Anti-UCHL1 from Cell Signaling Technology (11896) was used for western blot; Anti-UCHL1 from R&D Systems (MAB6007) was used for immunoprecipitation. Additional antibodies used for western blot were anti-cyclin D1 (sc-753, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-cyclin E (sc-481, Santa Cruz Biotechnology Inc.), anti-p53 (48818, Cell Signaling Technology, Danvers, MA, USA), anti-ubiquitin (3936, Cell Signaling Technology), anti-β-actin (4967, Cell Signaling Technology), anti-β-catenin (9562, Cell Signaling Technology), anti-GSK3α/β (5676, Cell Signaling Technology), anti-p21 (sc-397, Santa Cruz Biotechnology Inc.), and anti-p27 (sc-529, Santa Cruz Biotechnology Inc.).
UCHL1 was transiently silenced by transfection with Silencer select siRNAs (s14616 and s14618; Life Technologies, Carlsbad, CA, USA) duplexed with Lipofectamine RNAiMAX (Life Technologies) at a final concentration of 5 nM. Non-targeting Silencer Select siRNA was the negative control (Life Technologies).

Kwan S.Y., Au-Yeung C.L., Yeung T.L., Rynne-Vidal A., Wong K.K., Risinger J.I., Lin H.K., Schmandt R.E., Yates M.S., Mok S.C, & Lu K.H. (2020). Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) Promotes Uterine Serous Cancer Cell Proliferation and Cell Cycle Progression. Cancers, 12(1), 118.

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Luciferase Assay for Hypoxia Signaling

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For luciferase assays, HeLa/5HRE‐Luc or HeLa/ ODD‐Luc cells were seeded in 96‐well plates at a concentration of 1 × 10⁵ cells/mL and incubated under normoxic conditions. After a 24‐hour incubation, cells were treated with each reagent for 1 hour. Cells were then transferred to normoxic or hypoxic conditions for another 24‐hour incubation and harvested in 100 μL of passive lysis buffer (Promega). Luciferase assays were performed using 100 μL of luciferase assay reagent (Promega) or dual luciferase assay kit (Promega) according to the manufacturer’s instructions. Western blotting analysis was performed using anti–HIF‐1α (BD Biosciences), anti–UCHL1 (R&D Systems), anti–α‐tubulin (Sigma‐Aldrich), anti–β‐actin (Sigma‐Aldrich) and anti–β‐tubulin (Abcam) as primary antibodies. Alkaline‐phosphatase conjugated goat anti–mouse IgG antibody (Promega) was used as the secondary antibody. 5‐Bromo‐4‐chloro‐3‐indolyl‐phosphate 4‐toluidine salt (BCIP) and nitroblue tetrazolium (NBT) (Nacalai Tesque) was used to detect the indicated proteins.

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