Anti upf1

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, Canada

Anti-UPF1 is a primary antibody that specifically recognizes the UPF1 protein. UPF1 is a key regulator of the nonsense-mediated mRNA decay (NMD) pathway, which is responsible for the degradation of mRNAs containing premature termination codons.

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Lab products found in correlation

Anti flag, by Merck Group (2 mentions) Mini protean tgx gel, by Bio-Rad (2 mentions) Ecl reagent, by Thermo Fisher Scientific (1 mentions) Anti β actin, by Santa Cruz Biotechnology (1 mentions) Anti plcγ, by Santa Cruz Biotechnology (1 mentions) Anti egfp, by Takara Bio (1 mentions) Anti gst, by Merck Group (1 mentions) Anti calnexin, by Santa Cruz Biotechnology (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Ab9106, by Abcam (1 mentions) Ab134566, by Abcam (1 mentions) Ab56416, by Abcam (1 mentions) Anti sp1 sc 14027, by Santa Cruz Biotechnology (1 mentions) Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions) Hyperfilm ecl, by Cytiva (1 mentions) Anti gapdh, by Cell Signaling Technology (1 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Anti β actin, by Merck Group (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Ab172730, by Abcam (1 mentions)

7 protocols using anti upf1

Western Blot Detection of Proteins

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Proteins in cell lysates were resolved by electrophoresis on 6–10% polyacrylamide gels and subsequently transferred to PVDF membranes (Millipore). The following antibodies were used: anti-EGFP (Clontech), anti-β-actin (Santa Cruz), anti-GST (Sigma), anti-UPF1 (Cell Signaling), anti-UPF2 (Santa Cruz Biotechnology), anti-calnexin (Santa Cruz Biotechnology), anti-REQ (in-house manufactured anti-rabbit peptide antibodies raised against the peptide Ac-LGEFPVSNSRARC-NH₂, Mimotopes) and anti-PLCγ (Santa Cruz Biotechnology). Antibodies were diluted 1:200–1:1500 in 5% non-fat dry milk in 0.1% Tween 20/TBS and then incubated overnight at 4°C with rocking. Immunoreactive bands were then visualized using ECL reagents (Thermo).

Kim M.Y., Park J., Lee J.J., Ha D.H., Kim J., Kim C.G., Hwang J, & Kim C.G. (2014). Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3′UTR. Nucleic Acids Research, 42(11), 6999-7011.

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Western Blot Analysis of Protein Expression

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Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with Halt protease inhibitor cocktail [Thermo Fisher Scientific]) to obtain whole-cell lysates or lysed using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Fisher Scientific) to obtain cytoplasmic and nuclear fractions. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Blots were incubated with the indicated primary antibody: anti-UPF3B (ab134566; Abcam), anti-UPF1 (12040; Cell Signaling Technology, Inc.), anti-ZIKV capsid (C) (GTX133304; GeneTex), anti-Flag (F7425; Sigma-Aldrich), anti-β-actin (A5316; Sigma-Aldrich), anti-ZIKV envelope (E) (GTX133314; GeneTex), anti-SP1 (sc-14027; Santa Cruz Biotechnology), anti-GAPDH (5174; Cell Signaling Technology, Inc.), anti-Myc tag (ab9106; Abcam), anti-Strep tag (ab18422, Abcam), and anti-p62 (ab56416, Abcam). Proteins were visualized by chemiluminescent detection with ECL and ECL Hyperfilm (Amersham). Differences in band intensity were quantified by densitometry using ImageJ.

Fontaine K.A., Leon K.E., Khalid M.M., Tomar S., Jimenez-Morales D., Dunlap M., Kaye J.A., Shah P.S., Finkbeiner S., Krogan N.J, & Ott M. (2018). The Cellular NMD Pathway Restricts Zika Virus Infection and Is Targeted by the Viral Capsid Protein. mBio, 9(6), e02126-18.

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Identifying LINC00689 RNA-Binding Proteins

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RIP was performed to confirm the potential RNA-binding proteins of LINC00689 by a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s protocol. Briefly, cell lysis was conducted using RIP lysis buffer containing RNase and proteinase inhibitor, and then incubated with anti-U2AF2 (1:50, #70471, Cell Signaling Technology, Beverly, MA, USA), anti-SRSF1 (1:150, #32-4500, Thermo Fisher Scientific, Waltham, MA, USA), anti-PTBP1 (1:50, #32-4800, Thermo Fisher Scientific), anti-UPF1 (1:50, #9435, Cell Signaling Technology), or anti-IGF2BP2 (1:100, ab128175, Abcam) controlled by normal rabbit IgG (1:100, ab172730, Abcam) at 4°C overnight. After treatment with proteinase K buffer, the immunoprecipitated RNAs were extracted using the RNeasy MinElute Cleanup Kit (Qiagen, Duesseldorf, Germany), and then the enrichment of LINC00689 was measured by RT-qPCR.

Cao Y., Li J., Zhang G., Fang H., Du Y, & Liang Y. (2024). KLF15 transcriptionally activates LINC00689 to inhibit colorectal cancer development. Communications Biology, 7, 130.

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Western Blot Analysis of Protein Targets

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Cells were lysed using RIPA buffer (R0278, Sigma-Aldrich, St. Louis, MO, USA). Extracted proteins separated by SDS-PAGE (1610174, Bio-Rad Laboratories, Shanghai, China) were moved to PVDF membrane (IPVH00010, Millipore, Bedford, MA, USA). After membranes were sealed with 5% nonfat milk, primary antibodies including Anti-NETO2 (ab109288, Abcam, Cambridge, MA, UK), Anti-YY1 (ab109228, Abcam), Anti-UPF1 (9435, Cell Signaling Technology, Danvers, MA, USA), and Anti-GAPDH (ab8245, Abcam) were added for incubation overnight at 4°C. GAPDH was used as an internal reference. Later, the membranes were incubated with secondary antibody at room temperature for 1 h. The western blots were finally subject to enhanced chemiluminescence detection.

Chen X., Xu W., Ma Z., Zhu J., Hu J., Li X, & Fu S. (2021). TTN-AS1 accelerates the growth and migration of nasopharyngeal carcinoma cells via targeting miR-876-5p/NETO2. Molecular Therapy Oncolytics, 24, 535-546.

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Protein Expression and Detection Protocol

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Proteins were resolved through SDS-PAGE using 4–15% precast Mini-PROTEAN TGX Gels (Bio-Rad) and NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific) before transferring to a PVDF membrane (Bio-Rad). Immunodetection was achieved with 1:5,000 anti-ZAP (Abcam, Cambridge, United Kingdom); 1:5,000 anti-TRIM25 (BD Biosciences), 1:1,000 anti-HA (Roche Life Science), 1:5000 anti-V5 (Invitrogen), 1:2,500 anti-myc (Cell Signaling Technology, Danvers, MA), 1:20,000 anti-FLAG (Sigma-Aldrich), 1:500 anti-G3BP1 (Santa Cruz, Dallas, TX), 1:1,000 anti-G3BP2 (Assay Biotech, Fremont, CA), 1:1,000 anti-UPF1 (Cell Signaling Technology), 1:10,000 anti-NME1 (Abcam), 1:2,000 anti-PABPC4 (Proteintech, Rosemont, IL), and 1:20,000 anti-actin-HRP (Sigma-Aldrich). Primary antibodies were detected with 1:20,000 goat anti-mouse HRP (Jackson ImmunoResearch, West Grove, PA), 1:20,000 goat anti-rabbit HRP (Thermo Fisher Scientific), or 1:20,000 donkey anti-rat HRP (Jackson ImmunoResearch). Proteins were resolved on a 4–15% Mini-PROTEAN TGX gel (Bio-Rad, Hercules, CA) and visualized using ProSignal Pico ECL Reagent (Genesee Scientific, San Diego, CA) on a ChemiDoc (Bio-Rad). Quantification of western blots was performed using ImageJ.

Yang E., Huang S., Jami-Alahmadi Y., McInerney G.M., Wohlschlegel J.A, & Li M.M. (2022). Elucidation of TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes. PLoS Pathogens, 18(9), e1010743.

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In Situ Proximity Ligation Assay

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The in situ proximity ligation assay (PLA) was performed on fixed and permeabilised cells using Duolink In Situ Red kit mouse/rabbit (Sigma-Aldrich, DUO92101), according to the manufacturer’s protocol. Briefly, fixed and permeabilised cells were incubated with a blocking solution for 1 h at 37 °C and then incubated with the following primary antibodies: anti-UPF1 (Cell Signaling Technology, 12040; 1:200) and LIN28A (Cell Signaling Technology, 5930; 1:200) overnight at 4 °C. After incubation, the coverslips were washed twice with 1× buffer A, followed by incubation with PLA probes for 1 h at 37 °C. After washing twice with 1× buffer A for 5 min, the coverslip was incubated with the ligation solution for 30 min at 37 °C. Coverslips were washed twice with 1× buffer A, followed by an amplification step with polymerase for 100 min at 37 °C. Finally, the coverslip was washed twice with 1× buffer B and once with 0.01× buffer B and then mounted in VECTASHIELD with DAPI mounting medium (Vector Laboratories, H1200).

Jung S., Ko S.H., Ahn N., Lee J., Park C.H, & Hwang J. (2024). Role of UPF1-LIN28A interaction during early differentiation of pluripotent stem cells. Nature Communications, 15, 158.

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Western Blot Analysis of Protein Samples

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Adjusted protein lysates were supplemented with loading buffer (12% glycerol, 60 mM Na₂EDTA pH 8, 0.6% SDS, 0.003% bromphenol blue) and denatured for 10 minutes at 95 °C before SDS-PAGE. Proteins were wet-transferred to a nitrocellulose membrane at 80 V for 2 hours in transfer buffer (25 mM Tris-HCl pH 7.6, 192 mM glycine, 20% methanol, 0.03% SDS). Membranes were blocked in 5% milk powder diluted in TBST (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.05% Tween 20), before an overnight incubation at 4 °C with the primary antibodies. After three times washing with TBST, membranes were incubated with HRP-coupled secondary antibodies for 1 hour at room temperature, followed by another three washes with TBST. Blots were developed with Pierce ECL Substrate Kit following the manufacturer’s instructions. The following antibodies were used: anti-FLAG M2 (mouse, F1804, Sigma-Aldrich), anti-GAPDH (mouse, ACR001P, Acris), anti-TRIM71 (rabbit, kind gift from the Yamanaka lab), anti-TRIM71 (rabbit, PAB19293, Abnova), anti-SHCBP1 (rabbit, 12672–1-AP, Proteintech), anti-Cdkn1a/p21 (rabbit, 2947, Cell Signaling Technology), anti-EGR1 (rabbit, 4154, Cell Signaling Technology), anti-UPF1 (rabbit, 9435, Cell Signaling Technology), anti-VINCULIN (mouse, V9131, Sigma-Aldrich), anti-ubiquitin (rabbit, 3933, Cell Signaling Technology) and anti-tubulin (mouse, T9026, Sigma-Aldrich).

Duy P.Q., Weise S.C., Marini C., Li X.J., Liang D., Dahl P.J., Ma S., Spajic A., Dong W., Juusola J., Kiziltug E., Kundishora A.J., Koundal S., Pedram M.Z., Torres-Fernández L.A., Händler K., De Domenico E., Becker M., Ulas T., Juranek S.A., Cuevas E., Hao L.T., Jux B., Sousa A.M., Liu F., Kim S.K., Li M., Yang Y., Takeo Y., Duque A., Nelson-Williams C., Ha Y., Selvaganesan K., Robert S.M., Singh A.K., Allington G., Furey C.G., Timberlake A.T., Reeves B.C., Smith H., Dunbar A., DeSpenza T J.r., Goto J., Marlier A., Moreno-De-Luca A., Yu X., Butler W.E., Carter B.S., Lake E.M., Constable R.T., Rakic P., Lin H., Deniz E., Benveniste H., Malvankar N.S., Estrada-Veras J.I., Walsh C.A., Alper S.L., Schultze J.L., Paeschke K., Doetzlhofer A., Wulczyn F.G., Jin S.C., Lifton R.P., Sestan N., Kolanus W, & Kahle K.T. (2022). Impaired neurogenesis alters brain biomechanics in a neuroprogenitor-based genetic subtype of congenital hydrocephalus. Nature neuroscience, 25(4), 458-473.

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