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Setdb1

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SETDB1 is a histone methyltransferase enzyme that catalyzes the trimethylation of histone H3 at lysine 9 (H3K9me3). It plays a role in the epigenetic regulation of gene expression.

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Lab products found in correlation

Mphosph8, by Proteintech (2 mentions) Pphln1, by Merck Group (2 mentions) Morc2, by Fortis Life Sciences (2 mentions) Dcaf1, by Proteintech (2 mentions) Flag tag (flag), by Novus Biologicals (2 mentions) Line 1 orf1p, by Merck Group (2 mentions) Flag tag (flag), by Merck Group (2 mentions) E cadherin, by Cell Signaling Technology (2 mentions) Gapdh, by Proteintech (2 mentions) N cadherin, by Cell Signaling Technology (1 mentions) Actin, by Merck Group (1 mentions) Histone h4, by Cell Signaling Technology (1 mentions) Brca1, by Fortis Life Sciences (1 mentions) Vinculin, by Santa Cruz Biotechnology (1 mentions) Histone h3, by Abcam (1 mentions) Rabbit hrp, by Jackson ImmunoResearch (1 mentions) Hsp60, by Merck Group (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Goat anti mouse hrp, by Thermo Fisher Scientific (1 mentions) Goat anti rabbit horseradish peroxidase hrp, by Jackson ImmunoResearch (1 mentions)

8 protocols using setdb1

1

Western Blot Analysis of Protein Markers

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Cells were washed in PBS, counted, normalized for cell number, and lysed directly in 1x SDS-PAGE sample buffer. Samples were run on NuPage 4–12% Bis-Tris gels followed by blotting onto nitrocellulose membranes. Primary antibodies used: FAM208A (Atlas, HPA00875), MPHOSPH8 (Proteintech, 16796–1-AP), PPHLN1 (Sigma, HPA038902), SETDB1 (Proteintech 11231–1-AP), DCAF1 (Proteintech, 11612–1-AP), FLAG (Novus, NB600–345), FLAG (Sigma, F1804, used for IP), MORC2 (Bethyl Labs, A300–149A), LINE-1 ORF1p (Millipore, MABC1152),and HA (Biolegend, 901501).
Yurkovetskiy L., Guney M.H., Kim K., Goh S.L., McCauley S., Dauphin A., Diehl W, & Luban J. (2018). Primate immunodeficiency virus Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex. Nature microbiology, 3(12), 1354-1361.
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2

Protein Extraction and Western Blotting in CRC Cells

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Total protein in CRC lines and subcutaneous tumors was extracted using RIPA lysis buffer (Boyotime, China) according to the reagent instructions. Western blotting was performed with the specific antibody, SETDB1(1:1000, Proteintech, 11231-1-AP), p21(1:1000, proteintech, 10355-1-AP), GAPDH (1:1000, Proteintech, 60004-1-Ig), E-cadherin (1:1000, Cell Signaling Technology, 3195 T), N-cadherin (1:1000, Cell Signaling Technology, 13116-T).
Cao N., Yu Y., Zhu H., Chen M., Chen P., Zhuo M., Mao Y., Li L., Zhao Q., Wu M, & Ye M. (2020). SETDB1 promotes the progression of colorectal cancer via epigenetically silencing p21 expression. Cell Death & Disease, 11(5), 351.
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3

Comprehensive Antibody Panel for Western Blotting

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Primary antibodies: Actin (Millipore, MAB1501, 1:10 000), DAXX (Sigma-Aldrich, D7810-2ML, 1:1000), DAXX (Cell Signaling Technology, #4533, 1:1000), ATRX (Abcam, ab97508, 1:1000), GAPDH (Cell Signaling Technology, #5174, 1:7000), RNase H1 (Abcam, ab229078, 1:1000), BRCA1 (Bethyl, A300-000A, 1:1000), GFP (Roche, 11814, 1:1000), Vinculin (Santa Cruz, sc-73614, 1:2000), HSP60 (Sigma-Aldrich, H3524, 1:2000), KAP1 (Abcam, ab10483,1:1000), SETDB1 (Proteintech,11231–1-AP,1:1500), Histone H3.3 (Abcam, ab176840, 1:1000), Histone H4 (Cell Signaling Technology, #13919, 1:1000). Secondary antibodies: HRP rabbit (Jackson Laboratory, Cat. No. 111-035-003, 1:50 000), HRP mouse (Amersham/Sigma, Cat. No. GENA931-1ML, 1:10 000).
Pinto L.M., Pailas A., Bondarchenko M., Sharma A.B., Neumann K., Rizzo A.J., Jeanty C., Nicot N., Racca C., Graham M.K., Naughton C., Liu Y., Chen C.L., Meakin P.J., Gilbert N., Britton S., Meeker A.K., Heaphy C.M., Larminat F, & Van Dyck E. (2023). DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks. Nucleic Acids Research, 52(3), 1136-1155.
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4

Western Blot Analysis of Protein Markers

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Cells were washed in PBS, counted, normalized for cell number, and lysed directly in 1x SDS-PAGE sample buffer. Samples were run on NuPage 4–12% Bis-Tris gels followed by blotting onto nitrocellulose membranes. Primary antibodies used: FAM208A (Atlas, HPA00875), MPHOSPH8 (Proteintech, 16796–1-AP), PPHLN1 (Sigma, HPA038902), SETDB1 (Proteintech 11231–1-AP), DCAF1 (Proteintech, 11612–1-AP), FLAG (Novus, NB600–345), FLAG (Sigma, F1804, used for IP), MORC2 (Bethyl Labs, A300–149A), LINE-1 ORF1p (Millipore, MABC1152),and HA (Biolegend, 901501).
Yurkovetskiy L., Guney M.H., Kim K., Goh S.L., McCauley S., Dauphin A., Diehl W, & Luban J. (2018). Primate immunodeficiency virus Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex. Nature microbiology, 3(12), 1354-1361.
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5

Western Blot Analysis of Epigenetic Regulators

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Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis and extraction buffer containing protease inhibitor for 20 min. The lysate was clarified by centrifugation at 4°C for 10 min at 12,000 rpm. The samples were boiled at 95°C in SDS sample buffer containing β-mercaptoethanol and resolved by SDS-PAGE electrophoresis. The resolved proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo semidry transfer system (Bio-Rad), and the membrane was blocked in 1× PBS containing 0.1% Tween 20 and 5% skim milk powder for 1 h at room temperature. The membranes were probed with specific primary and secondary antibodies. The primary antibodies used were mouse anti-actin (catalog no. ab3280) and PPHLN1 (catalog no. ab69569), which were purchased from Abcam. SETDB1 (catalog no. 11231-1-AP) and MPP8 (catalog no. 16796-1-AP) were purchased from Proteintech. ZNF638/NP220 (catalog no. A301-548A) was purchased from Bethyl Laboratories; TASOR (catalog no. HPA006735) was from Sigma-Aldrich. Goat anti-rabbit-horseradish peroxidase (HRP) (catalog no. 111-035-003) was obtained from Jackson ImmunoResearch. Goat anti-mouse-HRP (catalog no. 32430) was obtained from Thermo Fisher.
Das A., Vijayan M., Walton E.M., Stafford V.G., Fiflis D.N, & Asokan A. (2022). Epigenetic Silencing of Recombinant Adeno-associated Virus Genomes by NP220 and the HUSH Complex. Journal of Virology, 96(4), e02039-21.
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6

Osteogenic Differentiation Protocol

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Alpha-modified essential medium (α-MEM) and trypsin/EDTA were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Penicillin–streptomycin solution was obtained from Beyotime Biotechnology (Shanghai, China). Dimethyl Sulfoxide (DMSO), insulin, dexamethasone, isobutyl methylxanthine (IBMX), and indomethacin were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Parathyroid hormone (hPTH1–34, H-4835) was from BACHEM Inc. (Torrance, CA, USA). LipofectamineTM 2000 was purchased from Invitrogen™ (Thermo Fisher Scientific). The antibody for Atf7ip was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The antibodies for H3, Runx2, Gapdh and Setdb1 came from Proteintech Group (Rosemont, IL, USA). The antibodies for H3K9me3, Sp7 and β-actin were acquired from Abcam (Cambridge, MA, USA).
Hu G., Shi X., Qu X., Han C., Hu A., Jia Z., Yang J., Liu H, & Wu Y. (2023). Atf7ip Inhibits Osteoblast Differentiation via Negative Regulation of the Sp7 Transcription Factor. International Journal of Molecular Sciences, 24(5), 4305.
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7

Epithelial-Mesenchymal Transition Markers

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The antibodies used included SLC38A3 (Proteintech, 14315-1-AP), E-cadherin (Cell Signaling Technology, Rabbit mAb#3195), Vimentin (Cell Signaling Technology, Rabbit mAb#5741), Snail (Cell Signaling Technology, Rabbit mAb#3879), Twist (Cell Signaling Technology, #46702), SETDB1 (Proteintech, 11231-1-AP), β-actin (Proteintech, 66009-1-Ig), and GAPDH (Proteintech, 60004-1-Ig).
Liu R., Hong R., Wang Y., Gong Y., Yeerken D., Yang D., Li J., Fan J., Chen J., Zhang W, & Zhan Q. (2020). Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma. Chinese Journal of Cancer Research, 32(5), 547-563.
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8

SETDB1 IHC Protocol in FFPE Tissue

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The FFPE blocks were collected and 5.0‐m paraffin sections were used for IHC. IHC was performed according to the antibody instructions by Envision two‐step method. Rabbit anti‐human polyclonal antibody SETDB1 (Proteintech Group) was diluted at 1:200. The positive control referred to the reagent instructions, and the negative control used PBS instead of primary antibody. The positive signal was localized to the nucleus. The scoring rules of SETDB1 IHC results refer to Shen X, et al.11 Less than 3 is divided into negative, greater than or equal to 3 is positive.
Wang C., Xia Z., Li Z., Ye F., Ji S., Lu C, & Zhang H. (2021). Expression of SET domain bifurcated histone lysine methyltransferase 1 and its clinical prognostic significance in hepatocellular carcinoma. Journal of Clinical Laboratory Analysis, 36(1), e24090.
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