Anti uchl1

Manufactured by Merck Group

Anti-UCHL1 is a laboratory reagent used for detecting the presence and expression levels of the UCHL1 protein in biological samples. UCHL1 is an enzyme involved in the regulation of protein function and turnover. This product can be utilized in various research applications that require analysis of UCHL1 protein levels.

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UCHL1 (2 citations)

3 protocols using anti uchl1

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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Detailed Lung Tissue Histology and IHC

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Lung lobes were inflated with 4% formalin and fixed for 24 hours, stored in 70% ethanol, paraffin-embedded, and 4 μm thick sections were used for Hematoxylin and Eosin (H&E) staining and immunohistochemistry (IHC). Primary antibodies were anti-RFP (Rockland, 600-401-379), anti-TTF1(Abcam, ab76013), anti-UCHL1(Sigma, HPA005993), anti-TP63 (Cell Signaling Technology, 13109), anti-phospho-S6 (Cell Signaling Technology, 4858), anti-PTEN (Cell Signaling Technology, 9559), anti-phospho-ERK (Cell Signaling Technology, 4370), anti-phospho-AKT (Thermo Fisher Scientific, 44-621G), and anti-HMHGA2 (Biocheck, 59170AP). IHC was performed using Avidin/Biotin Blocking Kit (Vector Laboratories, SP-2001), Avidin-Biotin Complex kit (Vector Laboratories, PK-4001), and DAB Peroxidase Substrate Kit (Vector Laboratories, SK-4100) following standard protocols.
To quantify the positivity of phospho-ERK and phospho-AKT stained slides, H-scores were calculated using Qupath. The H-score is determined by adding the results of multiplication of the percentage of cells with staining intensity ordinal value (scored from 0 for “no signal” to 3 for “strong signal”) with possible values ranging from 0 to 300 (37 (link)). To normalize potential variations between different rounds of immunohistochemistry, one patient sample was included and stained for both pERK and pAKT in all rounds of staining as a control.

Yousefi M., Boross G., Weiss C., Murray C.W., Hebert J.D., Cai H., Ashkin E.L., Karmakar S., Andrejka L., Chen L., Wang M., Tsai M.K., Lin W.Y., Li C., Yakhchalian P., Colón C.I., Chew S.K., Chu P., Swanton C., Kunder C.A., Petrov D.A, & Winslow M.M. (2022). Combinatorial inactivation of tumor suppressors efficiently initiates lung adenocarcinoma with therapeutic vulnerabilities. Cancer research, 82(8), 1589-1602.

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Tissue Fixation and Immunohistochemistry

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Lung lobes were inflated with 4% formalin and fixed for 24 h, stored in 70% ethanol, and then paraffin-embedded. We used 4-μm-thick sections for hematoxylin and eosin staining and IHC. Anti-RFP (Rockland, 600-401-379), anti-TTF1(Abcam, ab76013), anti-UCHL1(Sigma, HPA005993), and anti-TP63 (Cell Signaling Technology, 13109) IHC was performed using the Avidin/Biotin Blocking Kit (Vector Laboratories, SP-2001), Avidin-Biotin Complex kit (Vector Laboratories, PK-4001), and DAB (3,3’-diaminobenzidine) Peroxidase Substrate Kit (Vector Laboratories, SK-4100) following standard protocols.

Yousefi M., Andrejka L., Szamecz M., Winslow M.M., Petrov D.A, & Boross G. (2023). Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma. Proceedings of the National Academy of Sciences of the United States of America, 120(38), e2303224120.

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