Western blot assay was performed following the protocol as previously described [24 (link), 25 (link)]. Briefly, the cells were harvested and lysed using ice-cold RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1% Nonidet P-40, and 0.5% sodium deoxycholate). Following centrifugation at 10,000×g for 15 min at 4 °C, the proteins in the supernatants were quantified by Bradford method and separated using 10% SDS-PAGE gel and electrotransferred from the gel to a nitrocellulose membrane (Merck & Co., Inc., Whitehouse Station, NJ, USA). Following blocking with 5% skimmed milk in phosphate-buffered saline, the membranes were immunoblotted with the primary antibodies, namely, CDK6 (GeneTex, San Antonio, Texas, USA), DOT1L, H3, H3K79me2, cyclin D3, and GAPDH (Abcam, Cambridge, UK) at 4 °C overnight. Specifically bound HRP-conjugated secondary antibodies were detected using an ECL detection system (ChemiDocTM XRS+ machine, Bio-Rad Laboratories). Protein levels of GAPDH and H3 were employed as loading controls. Densitometric analyses were performed using ImageJ software. Relative quantification was carried out after normalization to the band intensities of GAPDH or H3. A Mann-Whitney test was performed to assess the difference of protein expression between groups. Each experiment was performed in triplicate and repeated at least 3 times.
Dot1l
Manufactured by Abcam
Sourced in United States
DOT1L is a methyltransferase enzyme that catalyzes the methylation of histone H3 at lysine 79 (H3K79). It plays a role in the regulation of gene expression and chromatin structure.
Automatically generated - may contain errors
Lab products found in correlation
H3k79me2, by Abcam (1 mentions)
Chemidoc xrs machine, by Bio-Rad (1 mentions)
Cyclin d3, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
H3k79me3, by Abcam (1 mentions)
Rna polymerase 2, by Merck Group (1 mentions)
H3k79me1, by Abcam (1 mentions)
H3k4me3, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Sds page, by Bio-Rad (1 mentions)
3 protocols using dot1l
1
Western Blot Analysis of Cell Lysates
2
SET Knockdown in Human Liver Cells
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The human liver (HL-7702, L-02 for short) cell line was purchased from the Institute of Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The reagents for cell culture were purchased from Gibco (Australia). The SET knockdown L-02 cells were previously constructed (Yang et al. 2012 (link)).
Reagents for cell culture, Western-blot analysis, chromatin precipitation siRNA transfection, and Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit were obtained from Thermo Scienti c (Rockford, IL, U.S.). Antibodies against SET and GAPDH were purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.).
Antibodies against p53, Dot1L, histone H3, mono-, di-and tri-methylation at histone H3K79 (H3K79me, H3K79me2 and H3K79me3) were purchased from Abcam (Cambridge, MA, USA). Reagents for reverse transcription and RT-PCR were obtained from TAKARA (Shanghai, China).
Reagents for cell culture, Western-blot analysis, chromatin precipitation siRNA transfection, and Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit were obtained from Thermo Scienti c (Rockford, IL, U.S.). Antibodies against SET and GAPDH were purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.).
Antibodies against p53, Dot1L, histone H3, mono-, di-and tri-methylation at histone H3K79 (H3K79me, H3K79me2 and H3K79me3) were purchased from Abcam (Cambridge, MA, USA). Reagents for reverse transcription and RT-PCR were obtained from TAKARA (Shanghai, China).
3
Quantitative Western Blotting Analysis
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Western blotting was performed, as previously described [26, 33] . MEL cells were collected after washing with ice-cold PBS, and then lysed in RIPA lysis buffer (Solarbio, Beijing, China) containing the protease inhibitor cocktail (Roche, Switzerland). An equal amount of protein for each lysate was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, USA), and then transferred to nitrocellulose membranes. Antibodies were against mouse-GAPDH (1:1000, Zhong Shan Jin Qiao, China), MLL (1:500, Santa Cruz Biotechnology, USA), DOT-1L (1:1000, Abcam, USA), H3K4me3 (1:1000, Abcam, USA), H3K79me1 (1:500, Abcam, USA), RNA polymerase II (1:1000, Sigma, USA) and Histone H3 and H4 (1:200, Bioss, China). SDS-PAGE followed by coomassie blue staining was used to quantify the mass of globin concentration, as described [1,5e7] . Quantification was performed with the software Image J.
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