Hrp conjugated rabbit anti actin

Manufactured by Cell Signaling Technology

HRP-conjugated rabbit anti-actin is a secondary antibody that recognizes and binds to actin, a highly conserved cytoskeletal protein found in all eukaryotic cells. The antibody is conjugated to horseradish peroxidase (HRP), an enzyme that can be used to catalyze colorimetric or chemiluminescent reactions for the detection of actin in western blotting and other immunoassays.

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4 protocols using hrp conjugated rabbit anti actin

Genetic Mouse Models for mTOR Signaling

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Vav-iCre (#008610), hCD2-iCre (#008520) Tsc1^fl/fl (#005680), and Rptor^fl/fl (#013188) mice were obtained from Jackson laboratory. Rabbit anti-pS6 235/236 (Cell Signaling, Danvers MA #4858, 1:1000), rabbit anti-pS6 240/244 (Cell Signaling #5364, 1:1000), mouse anti-total S6 (Cell Signaling #2317, 1:500), rabbit anti-p4E-BP1 Ser65 (Cell Signaling #9456, 1:500), rabbit anti-4E-BP1 rabbit anti-alpha globin (Epitomics, Burlingame, CA #EPR3608, 1:5000), rabbit anti-neuron-specific enolase (Immunostar, Hudson, WI #22521, 1:100), and HRP-conjugated rabbit anti-actin (Cell Signaling #5125, 1:2500) were used for immunoblotting. KU-0063794 was from Selleckchem (Houston, TX), MLN0128 was from Active Biochem (Maplewood, NJ), phenylhydrazine was from Sigma (St. Louis, MO), and rapamycin was from LC Labs (Woburn, MA).

Knight Z.A., Schmidt S.F., Birsoy K., Tan K, & Friedman J.M. (2014). A critical role for mTORC1 in erythropoiesis and anemia. eLife, 3, e01913.

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Western blot analysis of protein modifications

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Western blots were carried out as described previously (Currais et al., 2015b (link)). The primary antibodies used were: HRP-conjugated rabbit anti-actin (#5125, 1/20,000), acetyl-histone H3 (Lys9) (#9649, 1/100000), phospho-ACC1 (#3661, 1/2000), total ACC1 (#4190, 1/1000), phospho-AMPK (#2535, 1/1000) and total AMPK (#2793, 1/1000), from Cell Signaling Technology; histone H3 (#ab24834, 1/100000) from Abcam. Horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, goat anti-mouse or rabbit anti-goat (BioRad) diluted 1/5000) were used.

Currais A., Huang L., Goldberg J., Petrascheck M., Ates G., Pinto-Duarte A., Shokhirev M.N., Schubert D, & Maher P. (2019). Elevating acetyl-CoA levels reduces aspects of brain aging. eLife, 8, e47866.

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Western Blot Analysis of Cellular Signaling Pathways

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For Western blotting, 300,000 MN9D cells were grown overnight in 35 mm
dishes prior to the indicated treatments. Total protein extracts were prepared
and analyzed by SDS-PAGE and Western blotting as described previously
[5 (link)]. The primary
antibodies used for Western blotting were: rabbit anti-phospho-Akt (ser473)
(#9271, 1/1000), rabbit anti-phospho-Stat6 (#9361, 1/1000),
rabbit anti-phospho-4E-BP1 (thr37/46) (#2855, 1/10,000), rabbit anti-Akt
(#9272, 1/10,000), rabbit anti-Stat6 (#9362, 1/1000), rabbit
anti-4E-BP1 (#9644, 1/250,000) and HRP-conjugated rabbit anti-actin
(#5125, 1/20,000) from Cell Signaling. Following washing, the Western
blots were incubated for 1 hr at room temperature in horseradish peroxidase-goat
anti-rabbit or goat anti-mouse (Biorad, Hercules, CA) diluted 1/2500 and
developed with the Super Signal reagent (Pierce, Rockford, IL). In all cases,
the same membrane was re-probed for actin and/or a parallel membrane was probed
with an antibody reacting with the total protein in order to provide a
normalization standard. Autoradiographs were scanned and analyzed using a Biorad
GS800 scanner. The experiments were repeated a minimum of three times with
independent protein samples.

Maher P, & Conti B. (2017). Deciphering the Pathways that Protect from IL-13-Mediated Potentiation of Oxidative Stress-Induced Dopaminergic Nerve Cell Death. Cytokine, 103, 114-120.

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Amyloid and Actin Immunoblotting

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Western blots were carried out as described previously^{27 (link)}. The primary antibodies used were: beta amyloid 6E10 (#803004, BioLegend); HRP-conjugated rabbit anti-actin (#5125, Cell Signaling Technology). Horseradish peroxidase-conjugated secondary goat anti-mouse (BioRad) antibody was used.

Huang L., McClatchy D.B., Maher P., Liang Z., Diedrich J.K., Soriano-Castell D., Goldberg J., Shokhirev M., Yates JR I.I.I., Schubert D, & Currais A. (2020). Intracellular amyloid toxicity induces oxytosis/ferroptosis regulated cell death. Cell Death & Disease, 11(10), 828.

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