Ab16049

Manufactured by Abcam

Sourced in United States

Ab16049 is a primary antibody produced in rabbit. It recognizes a specific target protein, but the exact protein target is not provided in the available information.

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Lab products found in correlation

6 protocols using ab16049

Immunoblotting of Dopaminergic Markers

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This was performed as previously described [20 (link)] with antibodies against D2R (1:500, rabbit), dopamine transporter (DAT; 1:500, rabbit), tyrosine hydroxylase (TH; 1:1000, rabbit) (AB5084P, AB1591P and AB152, Merck Millipore, Billerica, MA, USA), signal transducer and activator of transcription 3α (STAT3α; 1:1000, rabbit), DNMT1 (1:1000, rabbit), DNMT3a (1:1000, rabbit) (nos. 8768, 5032 and 3598; Cell Signaling Technology, Tokyo, Japan), DNMT3b (1 μg/ml, rabbit), ERRγ (1:1000, rabbit) and β-actin (1:10,000, mouse) (ab16049, ab128930 and ab6276; Abcam, Cambridge, MA, USA).

Kozuka C., Kaname T., Shimizu-Okabe C., Takayama C., Tsutsui M., Matsushita M., Abe K, & Masuzaki H. (2017). Impact of brown rice-specific γ-oryzanol on epigenetic modulation of dopamine D2 receptors in brain striatum in high-fat-diet-induced obesity in mice. Diabetologia, 60(8), 1502-1511.

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Fucoidan-Mediated Protein Regulation

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After cells were treated with fucoidan for 48 h, cell lysates were separated by electrophoresis on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After gel transference to polyvinylidene fluoride membranes (Millipore), the membranes were incubated with antibodies against E-cadherin (sc-8426, Santa Cruz, CA, USA), occludin (ab31721, Abcam, Cambridge, MA, USA), N-cadherin (sc-8424, Santa Cruz, CA, USA), vimentin (sc-6260, Santa Cruz, CA, USA), ADAM12 (ab16049, Abcam), TGF-βR2 (sc-220, Santa Cruz, CA, USA), PTEN (sc-7974, Santa Cruz, CA, USA), p-phosphoinositide 3-kinase (PI3K; ab182651, Abcam), t-PI3K (ab86714, Abcam), p-AKT (sc-16646, Santa Cruz, CA, USA), t-AKT (sc-8312, Santa Cruz, CA, USA), and glyceraldehyde-3-phosphate dehydrogenase (sc-25778, Santa Cruz, CA, USA). Subsequently, the membranes were incubated with suitable secondary antibodies. Bands for specific molecules were detected using enhanced chemiluminescence (ECL; Millipore).

Wu S.Y., Yan M.D., Wu A.T., Yuan K.S, & Liu S.H. (2016). Brown Seaweed Fucoidan Inhibits Cancer Progression by Dual Regulation of mir-29c/ADAM12 and miR-17-5p/PTEN Axes in Human Breast Cancer Cells. Journal of Cancer, 7(15), 2408-2419.

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Epigenetic Regulation Immunoprecipitation Protocol

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For immunoprecipitation, cells were lysed in RIPA buffer directly followed by sonication. Clarified cell lysates were incubated with the indicated antibodies overnight, and protein A/G beads (Santa Cruz Biotechnology, Inc.) were added for 3–5 h. Beads were washed four times with RIPA buffer. Proteins were eluted in SDS-sample buffer and subjected to western blot analyses. Band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
The following antibodies were used: LC3B (L7543; Sigma), p62 (M162-3B; MBL International Corporation, Woburn, MA, USA), DNMT1 (ab13537; Abcam), DNMT3A (ab2850; Abcam), DNMT3B (ab16049; Abcam), HDAC1 (sc7872; Santa Cruz Biotechnology Inc.), HDAC2 (sc7899; Santa Cruz Biotechnology Inc.), HDAC3 (sc376957; Santa Cruz Biotechnology Inc.), NCoR (ab24552; Abcam), acetyled Histone H3 (06-599; Sigma-Aldrich), Histone H3 (ab1791; Abcam), Histone H3K9ac (07-352; Sigma-Aldrich), Histone H3K4me3 (39915; Active Motif), Histone H3K27me3 (ab6002; Abcam), MeCP2 (ab2828; Abcam), and β-actin (A5441; Sigma).

Lee M., Nam H.Y., Kang H.B., Lee W.H., Lee G.H., Sung G.J., Han M.W., Cho K.J., Chang E.J., Choi K.C., Kim S.W, & Kim S.Y. (2021). Epigenetic regulation of p62/SQSTM1 overcomes the radioresistance of head and neck cancer cells via autophagy-dependent senescence induction. Cell Death & Disease, 12(3), 250.

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Quantification of DNA Methylation Regulators

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Cells were washed twice with cold phosphate-buffered saline (PBS) and lysed at 4°C in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 1% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA), 137 mM NaCl, 10% glycerol, and protease and phosphatase inhibitors (Roche, Basel, Switzerland). Proteins were separated by SDS-PAGE using the NuPAGE SDS-PAGE Gel System (Invitrogen) under reducing conditions. A total of 15 μg of protein was mixed with 9 μL of NuPage LDS sample buffer (4×) (Invitrogen, NP0007), 3.6 μL of NUPAGE Sample Reducing (Invitrogen, NP0009) and filled up to 36 μL with RIPA buffer. This mixture was incubated at 85°C for 10 min while shaking at 900 rpm. Samples were loaded on NuPage precast gels (Invitrogen). Membranes were probed with the following antibodies: SOX9 (GTX109661, GeneTex, Hsinchu City, Taiwan); DNMT1 (ab13537, Abcam, Cambridge, UK); DNMT3a (ab2850, Abcam, Cambridge, UK); DNMT3b (ab16049, Abcam, Cambridge, UK); GAPDH (ab9483, Abcam, Cambridge, UK);

Cheng P.F., Shakhova O., Widmer D.S., Eichhoff O.M., Zingg D., Frommel S.C., Belloni B., Raaijmakers M.I., Goldinger S.M., Santoro R., Hemmi S., Sommer L., Dummer R, & Levesque M.P. (2015). Methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognostic factor in advanced melanoma. Genome Biology, 16(1), 42.

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Western Blotting of DNMT3A and DNMT3B

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Western blotting was performed using the conventional method (Fukuda et al., 2021 (link); Liao et al., 2015 (link)). Briefly, proteins from hPSCs were extracted using RIPA lysis and extraction buffer (Thermo Fisher Scientific). The extracted proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 7.5% TGX gel (Bio-Rad, Hercules, CA, USA) for DNMT3B (ab16049; Abcam, Cambridge, UK) and 4%–20% TGX gel (Bio-Rad) for DNMT3A (2160S; Cell Signaling Technology, Danvers, MA, USA) and GAPDH (2118; Cell Signaling Technology).

Motosugi N., Sugiyama A., Okada C., Otomo A., Umezawa A., Akutsu H., Hadano S, & Fukuda A. (2022). De-erosion of X chromosome dosage compensation by the editing of XIST regulatory regions restores the differentiation potential in hPSCs. Cell Reports Methods, 2(12), 100352.

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Western Blot Analysis of Epigenetic Regulators

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Total cell extracts or different cellular fractions were loaded on a NuPAGE gel (4 to 12%, NP0321, Thermo Fisher Scientific) and transferred to a Nitrocellulose Blotting Membrane (10600016, Life Sciences). The following primary antibodies were used: CDCA7 (1:500; 15249-1-AP, Proteintech), DNMT3A (1:1000; ab13888, Abcam), DNMT3B (1:1000; ab16049, Abcam), HELLS (1:1000; 11955-1-AP, Proteintech), UHRF1 (1:1000; 12387, Cell Signaling Technology), DNMT1 (1:1000; 5032, Cell Signaling Technology), Tubulin (1:2000; T6199, Sigma-Aldrich), Vinculin (1:2000; V9131, Merck), and H3 (96C10, 3638, Cell Signaling Technology). Donkey anti-Rabbit 800CW (1:5000; 926-32213, Li-Cor), Donkey anti-mouse 680RD (1:5000; 926-68072, Li-Cor), Donkey anti-Mouse 800CW (1:5000; 926-32212, Li-Cor), and Goat anti-Rabbit 680RD (1:5000; 926-68071, Li-Cor) were used as secondary antibodies. Membranes were analyzed using Odyssey (Westburg).

Vukic M., Chouaref J., Della Chiara V., Dogan S., Ratner F., Hogenboom J.Z., Epp T.A., Chawengsaksophak K., Vonk K.K., Breukel C., Ariyurek Y., San Leon Granado D., Kloet S.L, & Daxinger L. (2024). CDCA7-associated global aberrant DNA hypomethylation translates to localized, tissue-specific transcriptional responses. Science Advances, 10(6), eadk3384.

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