Characterization of Histone Methylation Markers by Western Blotting and Immunofluorescence

For western blotting, antibodies specific for histone H3 (07-690, EMD Millipore), H3K9me3 (2F3)^{49 (link)}, H3K9me2 (6D11^{50 (link)} and 39754, Active Motif), histone H3 for Supplementary Fig. 2g (96C10, Cell Signaling), SETDB1 (Cp10377, Cell Applications), SUV39H1 (#8729, CST), SUV39H2 (LS-116360, LSBio), G9a (A8620A)^{7 (link)} and GLP (B0422B)^{7 (link)} were used as primary antibody. For histone proteins, IRDye 800CW Goat anti-Mouse IgG (926-32210, LI-COR) and IRDye 680RD Goat anti-Rabbit IgG (926-68071, LI-COR) were used as secondary antibody. For non-histone proteins, HRP-linked anti-Rabbit IgG (NA934, GE Healthcare) and HRP-linked anti-Mouse IgG (NA931, GE Healthcare) were used as secondary antibody. For immunofluorescence analysis, H3K9me2 (6D11), H3K9me3 (39161, Active Motif) were used for primary antibody, and Goat anti-Mouse IgG Alexa Fluor 568 (A-11031, Invitrogen) and Goat anti-Rabbit IgG Alexa Fluor 488 (A-11034) were used for secondary antibody. For ChIP analysis, antibodies specific for H3K9me3 (2F3) and H3K9me2 (6D11) were used. Specificity of antibodies for H3K9me2 and H3K9me3 were previously reported^{49 (link),50 (link)}.

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Fukuda K., Shimura C., Miura H., Tanigawa A., Suzuki T., Dohmae N., Hiratani I, & Shinkai Y. (2021). Regulation of mammalian 3D genome organization and histone H3K9 dimethylation by H3K9 methyltransferases. Communications Biology, 4, 571.

Publication 2021

IRDye 800CW Goat anti-Mouse IgG IRDye 680RD Goat anti-Rabbit IgG NA931 Goat anti-Mouse IgG Alexa Fluor 568 Goat anti-Rabbit IgG Alexa Fluor 488

Alexa 568 Alexa fluor 488 Anti igg Antibodies Antibodies specificity Antibody Cell Chip Goat Histone Histone h3 Immunofluorescence Irdye 800cw Mouse Proteins Rabbit Setdb1

Corresponding Organization :

Other organizations : RIKEN Center for Biosystems Dynamics Research, RIKEN Center for Sustainable Resource Science

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Variable analysis

independent variables

Antibodies specific for histone H3 (07-690, EMD Millipore)
H3K9me3 (2F3)
H3K9me2 (6D11 and 39754, Active Motif)
Histone H3 for Supplementary Fig. 2g (96C10, Cell Signaling)
SETDB1 (Cp10377, Cell Applications)
SUV39H1 (#8729, CST)
SUV39H2 (LS-116360, LSBio)
G9a (A8620A)
GLP (B0422B)

dependent variables

Histone protein levels measured by western blotting
H3K9me2 and H3K9me3 levels measured by immunofluorescence analysis
H3K9me2 and H3K9me3 levels measured by ChIP analysis

control variables

Histone H3 levels for western blotting (96C10, Cell Signaling)

controls

Positive controls: Not explicitly mentioned.
Negative controls: Not explicitly mentioned.

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