Ubiquitin

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Ubiquitin is a small, highly conserved protein found in all eukaryotic cells. It functions as a post-translational modification, covalently attaching to target proteins and marking them for degradation or other cellular processes.

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Anti-ubiquitin antibody (41 citations) Mouse anti-ubiquitin (18 citations) Ubiquitin (P4D1) (17 citations) K63-ubiquitin (6 citations) Ubiquitin (3933) (2 citations) Anti-ubiquitin antibody 3933 (2 citations) Ubiquitin (ab7254) (2 citations) Rabbit polyclonal anti-ubiquitin (2 citations) Rabbit anti-ubiquitin antibody (2 citations) α-ubiquitin (P4D1; (2 citations)

177 protocols using ubiquitin

Screening Compound Library Inhibitor Assay

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The screened compound library (L1400) was a product of Selleck Chemicals (Houston, TX). The inhibitors, including Necrostatin-1, GSK’872, z-VAD-fmk, MG132, SB216763, PD98059, and LY294002, were purchased from Selleck Chemicals. Cell culture medium, Fetal Bovine Serum (FBS), and supplements were purchased from Invitrogen (Grand Island, NY). Primary antibodies against EGFR (#4267, 1:1000), ERK1/2 (#9102, 1:1000), Akt (#4691, 1:1000), p-EGFR-Tyr1068 (#3777, 1:1000), p-ERK1/2-Thr202/Tyr204 (#4370, 1:1000), p-Akt-Ser473 (#4060, 1:1000), PARP (#9532, 1:1000), cleaved-caspase 3 (#9664, 1:1000), Bax (#14796, 1:1000), VDAC1 (#4661, 1:2000), cytochrome C (#11940, 1:1000), Mcl-1 (#39224, 1:1000), ubiquitin (#3936, 1:1000), ubiquitin (#43124, 1:1000), α-Tubulin (#2125, 1:5000), GSK3β (#12456, 1:1000), and β-actin (#3700, 1:10000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Flag-tag (F3165, 1:5000) antibody was obtained from Sigma Aldrich (St. Louis, MO). Antibodies against FBW7 (ab109617, 1:1000) and FBW7 (ab187815, 1:1000) were obtained from Abcam (Cambridge, UK). Antibodies for immunohistochemistry staining (IHC), including anti-ki67 (ab15580, 1:300) and anti-p-Akt (ab81283, 1:100) were obtained from Abcam. Anti-p-EGFR (#3777, 1:200), and anti-Mcl-1 (#39224, 1:100) were purchased from Cell Signaling Technology, Inc.

Yu X., Gao F., Li W., Zhou L., Liu W, & Li M. (2020). Formononetin inhibits tumor growth by suppression of EGFR-Akt-Mcl-1 axis in non-small cell lung cancer. Journal of Experimental & Clinical Cancer Research : CR, 39, 62.

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Antibody Validation for Protein Analysis

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Rabbit polyclonal anti-human MIIP antibody (HPA044948) was purchased from Sigma-Aldrich (St Louis, USA). The ITGB3 (#13166), ITGAV (#4711), β-catenin (#8480), FAK (#13009), p-FAK(Tyr 576/577, #3281), p-FAK(Tyr 397, #8556), AKT(#4691), p-AKT(Ser 473, #9271), E-cadherin (#3195), N-cadherin (#13116), Vimentin (#5741), Claudin-1(#13255), Myc-tag (#2278, #2276 S), Ubiquitin (#3933), and the HRP-conjugated goat anti-rabbit antibodies were obtained from Cell Signaling Technology (Beverly, USA). The VEGFA antibody (ab46154) was purchased from Abcam (Cambridge, UK). The HA antibody (#11867423001) was from Roche (Basel, Switzerland). The HRP-conjugated β-actin (HRP-66009) and GAPDH (HRP-60004) antibodies were obtained from Proteintech (Chicago, USA). The HRP-conjugated goat anti-mouse IgG were purchased from ZSGB-BIO (Beijing, China) respectively.

Gao Y., Fang Y., Huang Y., Ma R., Chen X., Wang F., Pei X., Gao Y., Chen X., Liu X., Shan J, & Li P. (2022). MIIP functions as a novel ligand for ITGB3 to inhibit angiogenesis and tumorigenesis of triple-negative breast cancer. Cell Death & Disease, 13(9), 810.

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Antibody Immunoblotting for Cellular Signaling

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We used the following antibodies and dilutions for this study: ubiquitin (catalogue (cat.) no. 3936S, Cell Signaling Technology; 1:20:00), ISG15 (cat. no. HPA004627, Sigma Aldrich/Merck; 1:1,000), GAPDH (cat. no. 2118, Cell Signaling Technology; 1:2,000), GFP trap beads (cat no. gta-100, ChromoTek), GFP (cat. no. sc-9996, Santa Cruz Biotechnology; 1:2,000), IRF3 (cat. no. 4302, Cell Signaling Technology; 1:2,000), phospho-IRF3(Ser396) (cat. no. 4947, Cell Signaling Technology; 1:1,000), IκBα (cat. no. 4812, Cell Signaling Technology; 1:2,000), phospho-IκBα(Ser32/36) (cat. no. 9246, Cell Signaling Technology; 1:1,000), TBK1 (cat. no. 3013, Cell Signaling Technology; 1:2,000), pTBK1 (cat. no. 3300-1 Epitomics; 1:1,000), NF-κB p65 (cat. no. 8008, Santa Cruz Biotechnology; 1:2,000), lamin B1 (cat. no. sc-373918, Santa Cruz Biotechnology; 1:2,000).

Shin D., Mukherjee R., Grewe D., Bojkova D., Baek K., Bhattacharya A., Schulz L., Widera M., Mehdipour A.R., Tascher G., Geurink P.P., Wilhelm A., van der Heden van Noort G.J., Ovaa H., Müller S., Knobeloch K.P., Rajalingam K., Schulman B.A., Cinatl J., Hummer G., Ciesek S, & Dikic I. (2020). Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature, 587(7835), 657-662.

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Immunoblotting of Soluble and Insoluble Proteins

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Whole cell extracts and immunoblotting were performed as described earlier (8 ). Soluble proteins were prepared by centrifugation of cell lysates for 15 min in a microfuge at 4 °C, 18,000g. To assay insoluble proteins, remaining cell pellets were resuspended in solubilization buffer (20 mM phosphate buffer pH 8.0, 300 mM NaCl, 2% SDS, 2 mM DTT, 1% Triton X-100, 8 M urea, and 1× protease inhibitor cocktail) and incubated at room temperature for 5 min, followed by centrifugation at 18,000g for 15 min at 4 °C in a microfuge. The supernatant was then heated with in sample buffer (Bio-Rad, Cat. No. 1610737) for 5 min at 37 °C. Antibodies recognizing the following epitopes were used for immunoblotting at the indicated dilutions: Acss2, 1:1000 (Cell Signaling Technology, Cat. No. 3658); GFP, 1:1000 (Invitrogen, Cat. No. A11121); α-tubulin, 1:5000 (Sigma, Cat. No. T9026); V5, 1:1000 (Invitrogen, Cat. No. R96025); LC3B, 1:1000 (Cell Signaling Technology, Cat. No. 2775); ubiquitin, 1:1000 (Cell Signaling Technology, Cat. No. 3936); HRP-linked anti-mouse IgG (Cell Signaling Technology, Cat. No. 7076); HRP-linked anti-rabbit IgG (Cell Signaling Technology, Cat. No. 7074).

Nagati J.S., Kobeissy P.H., Nguyen M.Q., Xu M., Garcia T., Comerford S.A., Hammer R.E, & Garcia J.A. (2021). Mammalian acetate-dependent acetyl CoA synthetase 2 contains multiple protein destabilization and masking elements. The Journal of Biological Chemistry, 297(3), 101037.

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Western Blot Antibody Protocol

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The antibodies used for Western blotting in this study were: hTERT (1531-1, clone Y182, Epitomics), SK2 (ab37977, Abcam), MKRN1 (ab72054, Abcam), rabbit V5 (ab9116, Abcam), Ubiquitin (3933S, Cell Signaling Technology), HA (3724, Cell Signaling Technology), Calnexin (Sc6465, Santa Cruz Biotechnology), Lamin B (Sc6216, Santa Cruz Biotechnology), mouse V5 (R96025, Invitrogen, USA).

Selvam S.P., De Palma R.M., Oaks J.J., Oleinik N., Peterson Y.K., Stahelin R.V., Skordalakes E., Ponnusamy S., Garrett-Mayer E., Smith C.D, & Ogretmen B. (2015). Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation. Science signaling, 8(381), ra58.

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Comprehensive Mitochondrial Protein Analysis

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Several primary antibodies were used: OPA1 (612607; BD Biosciences), PDH (ab110333; Abcam), GAPDH (MA5-15738; Thermo), HSP60 (12165; Cell Signaling), TOM20 (11802-1-AP; Proteintech), MPC1 (14462; Cell Signaling Technology), MPC2 (46141; Cell Signaling Technology), CS (14309; Cell Signaling Technology), ACO2 (6571; Cell Signaling Technology), DRP1 (611113; BD Biosciences), MFF (Gandre-Babbe and van der Bliek, 2008 (link)), IDH2 (56439; Cell Signaling Technology), Fumarase (4567; Cell Signaling Technology), SDHA (11998; Cell Signaling Technology), SDHB (ab14714; Abcam), SDHC (14575-1-AP; Proteintech), SDHD (6847; ProSci), ubiquitin (3933S; Cell Signaling Technology), and p62 (GP62-C; Progen). Secondary antibodies were purchased from Invitrogen: Alexa 488 anti-Rabbit IgG (A21206), Alexa 488 anti-Mouse IgG (A21202), Alexa 568 anti-mouse IgG (A10037), and Alexa 647 anti-mouse IgG (A31571).

Yamada T., Murata D., Kleiner D.E., Anders R., Rosenberg A.Z., Kaplan J., Hamilton J.P., Aghajan M., Levi M., Wang N.Y., Dawson T.M., Yanagawa T., Powers A.F., Iijima M, & Sesaki H. (2022). Prevention and regression of megamitochondria and steatosis by blocking mitochondrial fusion in the liver. iScience, 25(4), 103996.

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Metabolic Reprogramming in Lung Cancer

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The normal human bronchial epithelioid cell (HBE) and the non-small-cell lung cancer cell lines including NCI-H1650, NCI-H1299, A549, NCI-H460, HCC4006, NCI-H1975, and NCI-H358 were obtained from ATCC and cultured with recommended culture medium. The primary antibodies for western blotting including anti-TRAF6 (#8028), hexokinase-1 (#2024), hexokinase-2 (#2867), GLUT1 (#12939), PKM2 (#4053), LDHA (#35 82), VDAC-1 (#4661), phosphor-Akt (#4060), Akt (#8596), phosphor-S6 (#4858), and ubiquitin (#58395) as well as the secondary anti-rabbit IgG HRP (#7074) were products of Cell Signaling Technology Inc. (Danvers, MA). β-Actin (A5316) was obtained from Sigma-Aldrich. In immunohistochemistry staining, the primary antibodies against hexokinase-2 (ab227198) and Ki67 (ab15580) were products of Abcam. TRAF6 shRNA#1 (TRCN0000007350) and shRNA#2 (TRCN0000007351) were purchased from the Sigma Mission shRNA library. The constitutively active Akt (CA-Akt) plasmid (Cat. #10841) and pLKO.1 GFP shRNA (Cat. #30323) were purchased from Addgene (Cambridge, MA, USA). Recombinant human insulin-like growth factor 1 (IGF-1) was a product of R&D (Cat. 291-G1-200). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA).

Feng L., Feng S., Nie Z., Deng Y., Xuan Y., Chen X., Lu Y., Liang L, & Chen Y. (2021). TRAF6 Promoted Tumor Glycolysis in Non-Small-Cell Lung Cancer by Activating the Akt-HIFα Pathway. BioMed Research International, 2021, 3431245.

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Fibroblast Cell Culture and TGF-β Signaling

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Human fetal lung fibroblast (MRC5) cell lines were propagated in EMEM media (Gibco, Rockville, IL, USA) with 10% FBS (Hyclone, Logan, UT, USA), and 1% penicillin/streptomycin antibiotic mix (Lonza, Allendale, NJ, USA). The cells were kept in a 37 °C humidified incubator with 5% carbon dioxide. Immobilized protein A/G beads, FN, α-SMA, TβRI, HA tag, USP11, V5 tag, TβRII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-SMAD2, phospho-SMAD3, total SMAD2, SMAD3, and ubiquitin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Bleomycin, leupeptin, CHX, MTX, and antibodies against Flag-tag and β-actin were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TGF-β1 was purchased from Invitrogen (Carlsbad, CA, USA). Proteasome inhibitor MG-132 was from Calbiochem (KGaA, Darmstadt, Germany). DAPI was purchased from ThermoFisher Scientific (Waltham, MA, USA). All materials in highest grades used in the experiments are commercially available.

Jacko A.M., Nan L., Li S., Tan J., Zhao J., Kass D.J, & Zhao Y. (2016). De-ubiquitinating enzyme, USP11, promotes transforming growth factor β-1 signaling through stabilization of transforming growth factor β receptor II. Cell Death & Disease, 7(11), e2474-.

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Investigating HMGA1-GRP75 Interaction in Cancer Progression

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Fetal bovine serum (FBS) and cell culture medium were purchased from HyClone. MG132 and kanamycin were purchased from Sigma. JNK IN8 was obtained from MedChem Express. Plasmids for overexpressing HMGA1 or GRP75 and HMGA1‐specific short hairpin RNA (shRNA) were designed and synthesized by GeneChem. Small interfering RNA (siRNA) oligonucleotides targeting HMGA1 or GRP75 were obtained from RiboBio. A cell counting kit‐8 (CCK8) kit was purchased from Bioss. A migration assay kit was obtained from BD Biosciences. Cell invasion assay chambers (24 wells) were purchased from Corning. Cycloheximide (CHX) and primary antibodies against HMGA1, GRP75, Ki67, Ubiquitin, JNK, P‐JNK (Thr183/Tyr185), c‐JUN, P‐c‐JUN (Ser63), P‐c‐JUN (Ser73), and GAPDH were purchased from Cell Signaling Technology. A Pierce coimmunoprecipitation kit was obtained from Thermo Fisher Scientific.

Qiao G., Wang R., Wang S., Tao S., Tan Q, & Jin H. (2021). GRP75‐mediated upregulation of HMGA1 stimulates stage I lung adenocarcinoma progression by activating JNK/c‐JUN signaling. Thoracic Cancer, 12(10), 1558-1569.

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Immunoprecipitation and Western Blot Analyses

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EEA1, Rab5, HA tag, GST tag, LSP1, Ubiquitin, HRS, STAM1, β-TrCP and FLAG antibodies were obtained from Cell Signaling Technology (Danvers, MA). Tetherin and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), LSP1 antibody was purchased from BD. CellTrace Far-Red DDAO-SE (C34553), and Lipofectamine 2000 were obtained from Life Technologies Corp. FITC-conjugated p24 GAG (6604665) and RD1-conjugated p24 GAG (6604667) antibodies were obtained from Beckman Coulter, Inc. (Brea, CA). Anti-GFP antibody (FITC) was obtained from Abcam (Cambridge, MA). Interferon-α was purchased from PBL Assay Science (Piscataway, NJ)

Kulkarni R., Jiang S., Birrane G, & Prasad A. (2020). Lymphocyte-Specific Protein 1 (LSP1) Regulates Bone Marrow Stromal Antigen 2 (BST-2)-Mediated Intracellular Trafficking of HIV-1 in Dendritic Cells. FEBS letters, 594(12), 1947-1959.

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