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Tubulin and flag

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TUBULIN and FLAG are laboratory reagents used in scientific research. TUBULIN is a protein that forms the structural components of cellular microtubules, which play a crucial role in various cellular processes. FLAG is a commonly used epitope tag that can be attached to proteins to facilitate their detection and purification. Both TUBULIN and FLAG are widely used in biochemical and cell biology studies, but a more detailed description cannot be provided while maintaining an unbiased and strictly factual approach.

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Lab products found in correlation

P akt, by Cell Signaling Technology (2 mentions) Phosphatase, by Santa Cruz Biotechnology (1 mentions) P mek1 2, by Cell Signaling Technology (1 mentions) P c raf, by Cell Signaling Technology (1 mentions) P erk1 2, by Cell Signaling Technology (1 mentions) Ripa buffer, by Merck Group (1 mentions) Protease, by Roche (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) P616 drp1, by Cell Signaling Technology (1 mentions) Senp2, by Santa Cruz Biotechnology (1 mentions) Oxphos rodent wb antibody cocktail, by Abcam (1 mentions) Gapdh, by Merck Group (1 mentions) Phospho gsk3β, by Cell Signaling Technology (1 mentions) Gapdh, by R&D Systems (1 mentions) Smad2 3, by BD (1 mentions) Nedd4, by Cell Signaling Technology (1 mentions) Sb431542, by Merck Group (1 mentions) P smad2, by Cell Signaling Technology (1 mentions) Snail, by Cell Signaling Technology (1 mentions) Actinomycin d, by Merck Group (1 mentions)

4 protocols using tubulin and flag

1

Dissecting Kinase Signaling Pathways

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Cell lysates were made in RIPA buffer (Sigma) for direct Western blotting or in a PNE buffer (PBS:H2O at 1 :1, 0.5% NP40, 5 mM EDTA, 5% glycerol) for immunoprecipitation, with both buffers supplemented with protease (Roche) and phosphatase (Santa Cruz Biotechnology) inhibitor cocktails. Western blots and immunoprecipitations were performed using the following antibodies : p-ERK1/2 (T202/Y204), p-MEK1/2 (S217/221), p-AKT (Thr308), p-CRAF (S338), total ERK1/2, MEK1/2, MEK1, MEK2, AKT, CRAF, DUSP4 and HA (Cell Signaling Technology), TUBULIN and FLAG (Sigma), BRAF (F-7), BRAF (C-19), p-MEK1 (Thr291), p-MEK1 (S222) (Santa Cruz Biotechnology) and p-MEK2 (S226) (US Biological). Western blot quantification was performed using NIH Image J. The three-dimension structures of MEK1 (3EQC) and PTEN mutants were modeled by the I-TASSER online server. Modeling the V600EBRAF-MUTMEK1 dimer interface was based on the crystal structure of the WTBRAF-WTMEK1 dimer (4MNE), the MEK1-KSR2 dimer (2Y4I), and the asymmetric, vemurafenib-bound, V600EBRAF dimer (3GO7). Protein structures were visualized using Pymol™
Moriceau G., Hugo W., Hong A., Shi H., Kong X., Yu C.C., Koya R.C., Samatar A.A., Khanlou N., Braun J., Ruchalski K., Seifert H., Larkin J., Dahlman K.B., Johnson D.B., Algazi A., Sosman J.A., Ribas A, & Lo R.S. (2015). Tunable-combinatorial Mechanisms of Acquired Resistance Limit the Efficacy of BRAF/MEK Co-targeting but Result in Melanoma Drug Addiction. Cancer cell, 27(2), 240-256.
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2

Quantification of Protein Expression in Islets

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Proteins were extracted from isolated islets or NIT-1 cells using RIPA lysis buffer. Proteins (15–20 μg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, Clifton, NJ, USA). After incubation with specific antibodies, the membrane bands were visualized by Amersham Imager 680 Blot and Gel Imagers (GE Healthcare Life Sciences, Marlborough, MA, USA). Antibodies against SENP2 (Santa Cruz Biotechnology Inc.), p616 DRP1 and DRP1 (Cell Signaling Technology, Danvers, MA, USA), GFP/YFP (Arigo Biolaboratories, Hsinchu City, Taiwan), GAPDH (Merck Millipore, Burlington, MA, USA), tubulin and Flag (Sigma-Aldrich) and OXPHOS Rodent WB antibody cocktail (Abcam, Cambridge, UK) were used for western blotting.
Nan J., Lee J.S., Moon J.H., Lee S.A., Park Y.J., Lee D.S., Chung S.S, & Park K.S. (2022). SENP2 regulates mitochondrial function and insulin secretion in pancreatic β cells. Experimental & Molecular Medicine, 54(1), 72-80.
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3

Antibody Characterization and Cell Signaling Assays

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Antibodies were obtained from sources indicated. TMEPAI (Abnova, CA), pSmad3, V5 epitope tag (Rockland, PA), Smad2/3 (BD Biosciences, CA), Smad 4 (SantaCruz biosciences, CA), Flag and Tubulin (Sigma, MO), GAPDH (R&D, MN), Actin and p27 (Epitomics, CA), pSmad2, Akt, pAkt, p21, phosphoGSK-3β, Snail, PTEN and Nedd4 (Cell Signaling, MA), Smad 7 (Imgenex, CA). Other reagents were obtained as indicated below. ProLong® Gold Antifade Reagent with DAPI, Fluorescein phalloidin (Lifetechnologies, NY), TGF-β (R&D, MN), LY294002 (Calbiochem, CA), H33258 (Bisbenzimide), actinomycin D, SB431542 and cycloheximide (Sigma, MO); doxycline (Clonetech, CA).
Singha P.K., Pandeswara S., Geng H., Lan R., Venkatachalam M.A, & Saikumar P. (2014). TGF-β induced TMEPAI/PMEPA1 inhibits canonical Smad signaling through R-Smad sequestration and promotes non-canonical PI3K/Akt signaling by reducing PTEN in triple negative breast cancer. Genes & Cancer, 5(9-10), 320-336.
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4

Protein Expression and Quantification

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Muscle samples were lyzed and protein concentration was determined as previously described 28 . Immunoblotting was performed using the following primary antibodies: Hsp72 and Hsp90 (Enzo Life Sciences, NY, USA (formerly SRESSGEN)), FLAG and ±-tubulin (Sigma Aldrich, St. Louis, MO, USA), OXPHOS cocktail (Abcam, Cambridge, UK) and GAPDH, phospho-AKT Ser 473, total AKT, Phospho-GSK-3±/² (Ser21/9), total GSK-3² (27C10), from (Cell Signalling, Danvers, USA). Quantification was performed using Quantity One 1-D Analysis Software (BioRad, Hercules, CA, USA).
Marshall J.P.S., Estevez E., Kammoun H.L., King E.J., Bruce C.R., Drew B.G., Qian H., Iliades P., Gregorevic P., Febbraio M.A, & Henstridge D.C. (2018). Skeletal muscle-specific overexpression of heat shock protein 72 improves skeletal muscle insulin-stimulated glucose uptake but does not alter whole body metabolism. Diabetes, obesity & metabolism, 20(8).
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