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2 protocols using alexa fluor 555 goat anti rabbit ab150078

1

Chromatin Modifications in MEF Cells

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All cell lines were cultured in DMEM medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Millipore-Sigma) in a humidified incubator with 5% CO2 at 37 °C.
Initial experiments were performed on primary MEFs derived from 13.5 days NM1 WT and KO embryos46 (link). In other experiments were used immortalized MEFs (ATCC® CRL-2752) or cell lines derived from these immortalized cells.
The antibodies against H3K9me3 (ab8898), H3K27ac (ab4729), H3K4me1 (ab8895), H3K4me3 (ab8580), H3K9ac (ab10812), H3 (ab1791), γH2AX (ab2893), p53-K370 (ab183544), PCAF (ab12188), Set1 (ab70378), GAPDH (ab8245), and nonspecific rabbit IgG isotype control (ab37415) were purchased from Abcam (Cambridge, MA, USA). The antibody against NM1 has been previously characterized2 (link). Alexa Fluor 555 Goat Anti-Rabbit (ab150078) and Alexa Fluor 488 Goat Anti-Mouse (ab150117) were used as secondary antibodies for immunofluorescence, and horseradish peroxidase (HRP)-fused Goat Anti-Rabbit (ab6721) and Rabbit Anti-Mouse (ab6728) secondary antibodies for western blottings were purchased from Abcam. Hoechst 43222 (H1399) and ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; P36931) were purchased from Invitrogen, Waltham, MA, USA. All antibodies have been used according to the manufacturers’ protocols.
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2

Culturing and Immunostaining HeLa Cells

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HeLa cells (ATCC® CCL-2) were cultured in DMEM with high glucose, 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin (all from Millipore-Sigma, Burlington, MA, USA), in a humidified incubator with 5% CO2 at 37 °C. For peptide treatment, 5 × 105 cells were seeded in 6-well plates with 2 mL of full-DMEM medium 24 h before the treatment. Next day, the medium was exchanged with pre-warmed DMEM medium supplemented with respective peptides and incubated for defined times. As a negative control, HeLa cells were incubated with pre-warmed DMEM medium supplemented with 1× PBS without any of the CPPs.
Antibodies against fibrillarin (ab4566), coilin (ab87913), Sc35 (ab11826), tubulin (ab195883), 58K (ab27043) and M6PR (ab124767) were all purchased from Abcam (Cambridge, MA, USA). Alexa Fluor 555 Goat Anti-Rabbit (ab150078) and Alexa Fluor 488 Goat Anti-Mouse (ab150117), used as secondary antibodies, were also purchased from Abcam. Hoechst 43222 (H1399) were purchased from Invitrogen (Waltham, MA, USA).
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