ChIP-seq was carried out and ChIP-seq data alignment, filtration, peak calling and assignment, and cross-sample comparison were performed as described36 (link). Mouse anti-Flag tag M2 (Sigma F1804, 5 μg), anti-H3K3me3 (ab9050, 5 μg), and anti-H3K27me3 (Millipore 07-449, 5 μg) were used.
Mouse anti flag tag m2
Manufactured by Merck Group
The Mouse anti-Flag tag M2 is a monoclonal antibody that recognizes the Flag epitope tag, a small peptide sequence that can be fused to recombinant proteins for detection and purification purposes. This antibody can be used in various immunological techniques to identify and isolate Flag-tagged proteins.
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2 protocols using mouse anti flag tag m2
1
ChIP-seq for histone modifications
2
Immunoprecipitation and Mass Spectrometry of GALNT6
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Cell lines stably expressing GALNT6-HA or mock vector were established as described previously (10) . Cells were lysed in 0.5% NP-40 immunoprecipitation lysis buffer at 4˚C for 30 min and were then incubated with anti-HA agarose beads (Sigma-Aldrich; Merck KGaA) at 4˚C for 16 h. After washing with the same buffer, the proteins bound to the beads were eluted with 1% sodium deoxycholate buffer. Subsequently, samples were prepared for LC-MS as described previously (33) .
Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 30 min at 4˚C and incubated with mouse anti-FLAG-tag M2 (Sigma-Aldrich; Merck KGaA), rat anti-HA-tag (Roche Diagnostics GmbH), rabbit anti-FLAG-tag (Sigma-Aldrich; Merck KGaA), mouse anti-Golgi-58k (Sigma-Aldrich; Merck KGaA), and rabbit anti-GALNT6 (10) antibodies. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescent images were obtained with Olympus IX71 and FV3000 microscopes (Olympus Corporation).
Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 30 min at 4˚C and incubated with mouse anti-FLAG-tag M2 (Sigma-Aldrich; Merck KGaA), rat anti-HA-tag (Roche Diagnostics GmbH), rabbit anti-FLAG-tag (Sigma-Aldrich; Merck KGaA), mouse anti-Golgi-58k (Sigma-Aldrich; Merck KGaA), and rabbit anti-GALNT6 (10) antibodies. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescent images were obtained with Olympus IX71 and FV3000 microscopes (Olympus Corporation).
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