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Anti β actin 13e5

Manufactured by Cell Signaling Technology
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Anti-β-actin (13E5) is an antibody product offered by Cell Signaling Technology. It is designed to detect the β-actin protein, which is a key structural component of the cytoskeleton in eukaryotic cells. The antibody can be used for various applications, such as Western blotting, immunoprecipitation, and immunofluorescence.

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Lab products found in correlation

Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (3 mentions) Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions) Cell lysis buffer, by Cell Signaling Technology (2 mentions) Erk1 erk2, by R&D Systems (2 mentions) Anti phospho erk1 erk2 t202 y204, by R&D Systems (2 mentions) Anti phospho egfr tyr1068, by Cell Signaling Technology (2 mentions) Anti akt, by Cell Signaling Technology (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Sds polyacrylamide gel electrophoresis, by Bio-Rad (1 mentions) Af1576, by R&D Systems (1 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) Anti cleaved parp, by Cell Signaling Technology (1 mentions) Blocking one solution, by Nacalai Tesque (1 mentions) Ifnar1 mice, by Jackson ImmunoResearch (1 mentions) C57bl 6 male mice, by Jackson ImmunoResearch (1 mentions) Poly 1 c, by InvivoGen (1 mentions) Antibody against α tubulin, by Merck Group (1 mentions) Anti gapdh gt239, by GeneTex (1 mentions) Anti β catenin d10a8, by Cell Signaling Technology (1 mentions)

19 protocols using anti β actin 13e5

1

Western Blot Analysis of Cellular Signaling

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Protein aliquots of 25 μg each were separated by SDS polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were washed three times and then incubated with Blocking One solution (Nacalai Tesque, Inc., Kyoto, Japan) for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies against anti-RET (D3D8R) (#14698), anti-phospho-RET (Tyr905) (#3221), anti-AKT (#9272), anti-phospho-AKT (Ser473) (#4060), anti-cleaved PARP (#5625), anti-cleaved caspase-3 (#9664), and anti-β-actin (13E5) (#4970) antibodies (1:1,000 dilution each; Cell Signaling Technology, Danvers, MA). Additional antibodies were also used including anti-human/mouse/rat extracellular signal-regulated kinase (ERK) 1/ERK2 (0.2 μg/mL) (AF1576) and anti-phospho-ERK1/ERK2 (T202/Y204) (0.1 μg/mL) (AF1018) from R&D Systems. The membranes were washed three times and then incubated for 1 hour at room temperature with species-specific horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate, an enhanced chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). Each experiment was performed independently at least three times.
Arai S., Kita K., Tanimoto A., Takeuchi S., Fukuda K., Sato H, & Yano S. (2017). In vitro and in vivo anti-tumor activity of alectinib in tumor cells with NCOA4-RET. Oncotarget, 8(43), 73766-73773.
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2

Murine cell differentiation protocol

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C57BL/6 male mice (6-8 weeks of age) and Ifnar1 −/− mice were from the Jackson Laboratory. All mice experiments were performed in accordance with guidelines from the University of California, Los Angeles, Institutional Animal Care and Use Committee. HEK293T and RAW264.7 cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. RXRαfl/fl (WT) and LysM-Cre +/RXRαfl/fl (Rxra−/−) mice bone marrows were overnight shipped from Dr Mercedes Ricote's Lab (Spain)13 (link). WT, Ifnar1 −/− and Rxra −/− BMMs were differentiated as described previously45 (link). WT and Rxra −/− F9 embryocarcinoma cells, as well as RXR-specific agonists AGN194204 and LG268 were obtained from Dr Peter Tontonoz's Lab (University of California, Los Angeles). HX531 was obtained from Dr Hiroyuki Kagechika's lab (Tokyo Medical and Dental University). 9cRA and 13cRA were purchased from Sigma-Aldrich. All compounds for in vitro treatment were solubilized in DMSO. PolyI:C and polydA:dT were from InvivoGen. Antibody against α-tubulin was from Sigma-Aldrich. Anti-VSV-G (P5D4) and anti-Oct1 (C-21) antibodies were from Santa Cruz Biotechnology. Anti-RXRα (D6G10), anti-β-Actin (13E5) and anti-β-Catenin (D10A8) were from Cell Signaling Technology. Anti-GAPDH (GT239) was from GeneTex.
Ma F., Liu S.Y., Razani B., Arora N., Li B., Kagechika H., Tontonoz P., Núñez V., Ricote M, & Cheng G. (2014). Retinoid X receptor α attenuates host antiviral response by suppressing type I interferon. Nature communications, 5, 5494.
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3

Comprehensive Cell Signaling Analysis

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Cell lysis, Western blotting and immunohistochemistry (IHC) was done as previously described (9 (link),10 (link)). The following antibodies were used for immunostaining and Western blotting: anti-CDK4 (D9G3E), anti-cyclin D1 (92G2) and anti-β-actin (13E5) (Cell Signaling Technology, Danvers, MA, USA). The receptor tyrosine kinase (RTK) array (Proteome Profiler Human Phospho-RTK Array Kit, R&D Systems, Minneapolis, MN, USA) was purchased and used according to the manufacturer’s recommended conditions as previously described (9 (link)). List of 49 RTKs measuring relative phosphorylation status on antibody arrays were shown in Appendix 2.
Hirai N., Hatanaka Y., Hatanaka K.C., Uno Y., Chiba S.I., Umekage Y., Minami Y., Okumura S., Ohsaki Y, & Sasaki T. (2021). Cyclin-dependent kinase 4 upregulation mediates acquired resistance of dabrafenib plus trametinib in BRAF V600E-mutated lung cancer. Translational Lung Cancer Research, 10(9), 3737-3744.
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4

Protein Expression Analysis in Cells

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Cells were homogenized in RIPA lysis buffer for protein extraction, supplemented with protease inhibitors (Thermo Scientific, USA). Denatured proteins were separated in 12% SDS–PAGE and then transferred onto nitrocellulose papers (Pall, USA). After blotting, nitrocellulose papers were incubated with specific antibodies. Primary antibodies: anti-ERα antibody D8H8, 1:2000 (Cell Signaling Technology, USA, #8644); anti-ERα F-10, 1:1000 (Santa Cruz Biotechnology, USA, #sc-8002), anti-ERα 1D5, 1:1000 (Invitrogen, USA, #MA5-13191), anti-ERα-36, 1:200 (Alpha Diagnostic International, USA, #ERA361-A); anti-β-actin 13E5, 1:2000 (Cell Signaling Technology, USA, #8457); anti-HDAC2, 1:1000 (Cell Signaling Technology, USA, #2540); anti-VDAC1 N-18 (Santa Cruz Biotechnology, USA, #sc-8828). Secondary antibodies (HRP conjugated): anti-rabbit, 1:5000 (Cell Signaling Technology, USA, #7074); anti-mouse, 1:2000 (Cytiva, USA, #RPN4201). Immunolabelling was visualized using ECL procedure (PerkinElmer, USA). Uncropped and unprocessed scans of the most important blots are supplied in the Source Data file. All blots derive from the same experiment and they were processed in parallel.
Strillacci A., Sansone P., Rajasekhar V.K., Turkekul M., Boyko V., Meng F., Houck-Loomis B., Brown D., Berger M.F., Hendrickson R.C., Chang Q., de Stanchina E., Pareja F., Reis-Filho J.S., Rajappachetty R.S., Del Priore I., Liu B., Cai Y., Penson A., Mastroleo C., Berishaj M., Borsetti F., Spisni E., Lyden D., Chandarlapaty S, & Bromberg J. (2022). ERα-LBD, an isoform of estrogen receptor alpha, promotes breast cancer proliferation and endocrine resistance. NPJ Breast Cancer, 8, 96.
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5

Immunoblotting Analysis of Patient-Derived Xenograft Tumors

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Patient‐derived xenograft tumor lysates were prepared using cell lysis buffer (Cell Signaling) and immunoblotting was carried out as previously described.18 All antibodies were purchased from commercial companies as follows: anti‐E‐cadherin, anti‐Vimentin, anti‐ZEB1, anti‐β‐actin (13E5) (Cell Signaling Technology), diluted at a ratio of 1:1000. Antigen‐antibody reaction bands were visualized with the SuperSignal West Dura Extended Duration Substrate, an ECL substrate (Pierce Biotechnology). Experiments were independently repeated at least three times.
Kita K., Fukuda K., Takahashi H., Tanimoto A., Nishiyama A., Arai S., Takeuchi S., Yamashita K., Ohtsubo K., Otani S., Yanagimura N., Suzuki C., Ikeda H., Tamura M., Matsumoto I, & Yano S. (2019). Patient‐derived xenograft models of non‐small cell lung cancer for evaluating targeted drug sensitivity and resistance. Cancer Science, 110(10), 3215-3224.
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6

Western Blot Analysis of Ferritin and Actin

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Cells were washed twice with PBS and then lysed with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40) in the presence of protease and phosphatase inhibitors. Prior to SDS-PAGE, cell lysates were re-suspended in SDS sample buffer (60 mM Tris–HCl, 1% SDS, 10% glycerol, 0.05% bromophenol blue, pH 6.8, with 2% β-mercaptoethanol). Samples were subjected to 10% SDS-PAGE (Bio-Rad, Hercules, CA) and transferred to PVDF membranes (Bio-Rad) for immunoblot. Transfer efficiency was determined by Ponceau S staining (Sigma-Aldrich, St. Louis, MO). PVDF membranes were incubated with blocking solution (TBS containing 0.1% Tween 20% and 5% BSA) and were probed with specific antibodies including anti-Ferritin heavy chain (B-12) (sc-376594, Santa Cruz Biotechnology, Dallas, TX) and anti-β-Actin (13E5) (cat# 4970, Cell Signaling Technology, Danvers, MA). Protein bands were detected using a chemiluminescence kit (Millipore, Billerica, MA).
Camarena V., Sant D.W., Huff T.C., Mustafi S., Muir R.K., Aron A.T., Chang C.J., Renslo A.R., Monje P.V, & Wang G. (2017). cAMP signaling regulates DNA hydroxymethylation by augmenting the intracellular labile ferrous iron pool. eLife, 6, e29750.
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7

Osteoclastogenesis Assay Protocol

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Cells were cultured in αMEM containing 2 mM L-glutamine (12561), 10% heat-inactivated FBS, 100 IU/ml Penicillin and 100 IU/ml Streptomycin (Life Technologies). qPCR primers were either designed or used as previously described and tested for their distinctive product sizes (Integrated DNA Technologies) (34 (link)). For a detailed list of primer constructs see supplemental table 1. CMG-1412 media was generated as previously described (35 (link)). IF antibodies and reagents: anti-KCa3.1 (P4997) (Sigma-Aldrich), anti-CD68 (FA-11) (Biolegend) (36 (link)), Phalloidin-FITC (Sigma-Aldrich), goat anti-rabbit AF-594 and goat anti-rat AF488 (life technologies), goat serum (Gemini-Bio products). Recombinant cytokines (mM-CSF, mTNF and mRANKL) were purchased from R&D Systems; inhibitors: TRAM-34, KN-93, cyclosporine A (CsA) (Sigma-Aldrich); Fluo-4-AM (life technologies); Western blot antibodies: anti-CREB (06-863) and anti-phospho-CREB (Ser133) (06-519) (Millipore) (14 (link)), anti-CaMKIV (H-5) (Santa Cruz Technologies) (37 (link)), anti-phospho-CaMKIV (Thr196, Thr200) (PA5-37504) (Thermo Fisher Scientific), anti-NFATc1 (7A6) (Thermo Fisher Scientific) (14 (link)), anti-β-actin (13E5) (Cell Signaling Technology)
Grössinger E.M., Kang M., Bouchareychas L., Sarin R., Haudenschild D.R., Borodinsky L.N, & Adamopoulos I.E. (2017). Ca2+-dependent regulation of NFATc1 via KCa3.1 in Inflammatory Osteoclastogenesis. Journal of immunology (Baltimore, Md. : 1950), 200(2), 749-757.
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8

Western Blot Analysis of Signaling Proteins

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Sodium dodesyl sulfate (SDS) polyacrylamide gels (Bio-Rad, Hercules, CA) were loaded with 40 μg total protein per lane; following electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad), which were incubated with Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, followed by overnight incubation at 4°C with anti-ALK (C26G7), anti-phospho-ALK (Tyr1604), anti-phospho-EGFR (Tyr1068), anti-STAT3(79D7), anti-phospho-STAT3 (Y705), anti-AKT, anti-phospho-AKT (Ser473), anti-ErbB4 (111B2), anti-phospho-ErbB4 (Tyr1284), anti-MET (25H2), anti-phospho-MET (Y1234/Y1235) (3D7), or anti-β-actin (13E5) antibodies (1:1,000 dilution each; Cell Signaling Technology, Danvers, MA), or with anti-human EGFR (1 μg/mL), anti-human/mouse/rat extracellular signal-regulated kinase (Erk)1/Erk2 (0.2 μg/mL), or anti-phospho-Erk1/Erk2 (T202/Y204) (0.1 μg/mL) antibodies (R&D Systems). After washing 3 times, the membranes were incubated for 1 h at room temperature with secondary antibodies (horseradish peroxidase-conjugated species-specific antibodies).
Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate Enhanced Chemiluminescent Substrate (Pierce, Osaka, Japan). Each experiment was independently carried out at least 3 times.
Tanimoto A., Yamada T., Nanjo S., Takeuchi S., Ebi H., Kita K., Matsumoto K, & Yano S. (2014). Receptor ligand-triggered resistance to alectinib and its circumvention by Hsp90 inhibition in EML4-ALK lung cancer cells. Oncotarget, 5(13), 4920-4928.
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9

Immunoblot Analysis of HER2 Signaling

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Total protein lysates (20 μg) were extracted using RIPA buffer and separated on SDS-PAGE gels (NuPAGE™ 4–12% Bis-Tris Protein Gels, Invitrogen) according to standard methods.
Membranes were probed using the following antibodies: anti-total HER2 Rabbit mAb (29D8, Cell Signalling #2165), anti-total HER2 Mouse mAb (CB11, Thermo Scientific #MA1–35720), anti-phospho-HER2 (Tyr1248, Cell Signalling #2247), anti-Cleaved PARP Asp214 human specific (Cell Signalling Technology #9541), anti-phospho-tyrosine (Cell Signaling Technology #8954), anti-β-Actin 13E5 (Cell Signalling Technology #4970) and Ubiquitin (P4D1, Cell Signaling Technology #3936S).
Li B.T., Michelini F., Misale S., Cocco E., Baldino L., Cai Y., Shifman S., Tu H.Y., Myers M.L., Xu C., Mattar M., Khodos I., Little M., Qeriqi B., Weitsman G., Wilhem C.J., Lalani A.S., Diala I., Freedman R.A., Lin N.U., Solit D.B., Berger M.F., Barber P.R., Ng T., Offin M., Isbell J.M., Jones D.R., Yu H.A., Thyparambil S., Liao W.L., Bhalkikar A., Cecchi F., Hyman D.M., Lewis J.S., Buonocore D.J., Ho A.L., Makker V., Reis-Filho J.S., Razavi P., Arcila M.E., Kris M.G., Poirier J.T., Shen R., Tsurutani J., Ulaner G.A., de Stanchina E., Rosen N., Rudin C.M, & Scaltriti M. (2020). HER2-mediated internalization of cytotoxic agents in ERBB2 amplified or mutant lung cancers. Cancer discovery, 10(5), 674-687.
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10

Signal Pathway Inhibition in A549 Cell Response to BJ501 Infection

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The primary antibodies used in the analysis were anti-NFκB p65, anti-phospho-NFκB p65 (Ser536), anti-STAT3, anti-phospho-STAT3 (Tyr705), anti-PI3K, anti-phospho-PI3K p85 (Tyr458)/p55 (Tyr199), anti-Akt, anti-phospho-Akt (Thr308), anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-SAPK/JNK, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-Erk1/2, anti-phospho-Erk1/2 (Thr202/Tyr204), anti-β-actin (13E5) and anti-rabbit IgG were purchased from Cell Signaling Technology, Western Chemiluminescent Horseradish Peroxidase Substrate was purchased from Millipore Corporation, Signal pathway inhibitor including Fludarabine (STAT1, 100 μM, CAT.NO. T1038), SCH772984 (MEK1/ERK1/ERK2, 100 μM, CAT.NO. T6066), MCC950 (NLRP3, 10 μM, CAT.NO. T3701), Ruxolitinib phosphate (JAK1/2, 100 μM, CAT.NO. T3043), SB203580 (p38 MAPK, 100 μM, CAT.NO. T1764), Hydroxychloroquine sulfate (TLR7/9, 100 μM, CAT.NO. T0951) and SP600125 (JNK1/2/3, 10 μM, CAT.NO. T3109) were purchased from TargetMol, PDTC (NFκB, 100 μM, CAT.NO. S3633) were purchased from SELLECK. For signal pathway inhibitor administration, A549 cells were pretreated with the inhibitor for 12 h and the supernatants were removed, followed by BJ501 infection for 24 h.
Wang K., Gong M., Zhao S., Lai C., Zhao L., Cheng S., Xia M., Li Y., Wang K., Sun H., Zhu P., Zhou Y., Ao Q, & Deng X. (2023). A novel lncRNA DFRV plays a dual function in influenza A virus infection. Frontiers in Microbiology, 14, 1171423.
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