Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, 0.5% Na-Deoxycholate, 0.1% SDS, and 1% NP-40), and an equal amount of protein was resolved using Nupage Novex 4–12% Bis-Tris Gel (Thermo Fisher Scientific) as previously described in ref. 22 (link). Primary antibodies used were β-ACTIN (ACTB) (1:5,000, #2228; Sigma), ATRX (1:1,000, sc-15408; Santa Cruz Biotechnology), Flag (1:1,000, F1804; Sigma), H3 (1:2,000, ab1791; Abcam), H3K36me2 (1:1,000, 2901; Cell Signaling), H3K36me3 (1:2,000, 61101; Active Motif), KDM2A (1:1,000, A301-476A; Bethyl Laboratories), SENP6 (1:500, HPA024376; Sigma-Aldrich), SMC5 (1:2000, A300-236A; Bethyl Laboratories), and TUBULIN (1:2,000, ab15246; Abcam). Secondary antibodies used were donkey anti-rabbit HRP (1:1,000, sc-2077; Santa Cruz Biotechnology), donkey anti-mouse HRP (1:1,000; sc-2096, Santa Cruz Biotechnology), and donkey anti-goat HRP (1:1,000, sc-2056, Santa Cruz Biotechnology). Antibody signal was detected using the ECL Western Blotting Substrate (W1015, Promega) and X-ray film (F-BX810, Phenix).
Sc 15408
Manufactured by Santa Cruz Biotechnology
Sc-15408 is a laboratory equipment product. It is a component used in various research and experimental applications. The core function of Sc-15408 is to facilitate specific laboratory procedures, but a detailed description without interpretations or extrapolations cannot be provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab1791, by Abcam (3 mentions)
Ab22553, by Abcam (2 mentions)
Ab26300, by Abcam (2 mentions)
Ab10483, by Abcam (2 mentions)
Ab8898, by Abcam (2 mentions)
Tub 2, by Merck Group (2 mentions)
Sc 7152, by Santa Cruz Biotechnology (2 mentions)
Abe154, by Merck Group (2 mentions)
D9564, by Merck Group (2 mentions)
A32754, by Thermo Fisher Scientific (2 mentions)
Widefield microscope celldiscoverer7 cd7, by Zeiss (2 mentions)
Ab214, by Abcam (2 mentions)
Cellytic nuclear extraction kit, by Merck Group (2 mentions)
β actin actb, by Merck Group (1 mentions)
H3k36me2, by Cell Signaling Technology (1 mentions)
H3k36me3, by Active Motif (1 mentions)
Donkey anti rabbit hrp, by Santa Cruz Biotechnology (1 mentions)
Sc 2077, by Santa Cruz Biotechnology (1 mentions)
Donkey anti mouse hrp, by Santa Cruz Biotechnology (1 mentions)
Sc 2096, by Santa Cruz Biotechnology (1 mentions)
9 protocols using sc 15408
1
Western Blot Analysis of Protein Targets
2
Chromatin Remodeling Protein Analysis
3
Immunofluorescence Staining of ATRX in 3D Cell Cultures
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800 C3H/10T1/2 cells were seeded per well in a 96 well plate (glass bottom culture plates, MatTek, PBK96G-1.5-5-F) and kept at 37-degree Celcius for 24 h. Cells were fixed with 4% formaldehyde in PBS for 10 min at room temperature. Cells were permeabilized with 0.2% digitonin (EMD Millipore, 300410) in PBS (Corning, 21-040-CV) for 10 min at room temperature. Next, the cells were incubated in 3% BSA (in PBS, Sigma, #9418) for blocking for 1 h at room temperature followed by incubation in primary antibody (ATRX, Santa Cruz, sc-15408, 1:200) at 4°C overnight, followed by secondary antibody (1:600, Invitrogen, A32754) incubation at room temperature for 1 h. Cells were washed three times with PBS for 5 min followed by DAPI staining (2 μg/ml in PBS, Sigma, D9564) for 5 min at room temperature. Mounting media (Vectashield, H-1000) was added immediately after DAPI staining. Cells were imaged using Widefield Microscope CellDiscoverer7 (CD7) automated widefield high-throughput system (Zeiss). Images were processed with ImageJ software (http://rsb.info.nih.gov/ij/ ). For the ATRX antibody, the ImageJ Brightness/Contrast was set as 30/112. For DAPI, the Brightness/Contrast was set as 37/123. All images were processed with the same parameter settings for each antibody.
4
Antibody-based Detection of DNA Damage Markers
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Primary antibodies used were rabbit antibodies against ATRX (sc-15408, Santa Cruz), H3.3 (09–838, Millipore Merck), phosphorylated H3.3S31 (ab2889, Abcam; Active Motif, 39637; H3.3S31ph) (Abcam; Active Motif), CHK1 Ser317 (12302, Cell Signaling Technology), CHK2 Thr68 (abe343, Cell Signaling Technology), CHK1 (NBP1–19210, Novus Biologicals) and CHK2 (05–649, Millipore Merck); mouse monoclonal antibodies against γH2AX Ser139 (05–636, Millipore Merck), and myc tag (05–724, Millipore Merck), tubulin (Roche), and human anti centromere CREST serum (34 (link)). CHK1 inhibitor UCN-01 (Millipore Merck) and SB218078 (Millipore Merck) were used at a concentration of 1 μM and 5 μM, respectively (36 (link)). CHK2 inhibitor II hydrate (Sigma Aldrich) was used at a final concentration of 250 μM.
5
Immunostaining for ATRX in Mouse Brains
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Mice were perfused and the brains fixed for 72 h with 4% paraformaldehyde in PBS and cryopreserved in 30% sucrose/PBS. Brains were flash frozen in Cryomatrix (Thermo Fisher Scientific) and sectioned as described previously (Ritchie et al., 2014 (link)). For immunostaining, antigen retrieval was performed by incubating slides in 10 mM sodium citrate at 95°C for 10 min. Cooled slides were washed and incubated overnight in anti-ATRX rabbit polyclonal antibody (Santa Cruz Biotechnology, SC-15408; 1:200, H-300) (Watson et al., 2013 (link)) diluted in 0.3% Triton-X100 in PBS. Sections were washed and incubated with goat anti-rabbit Alexa Fluor 594 (Life Technologies) for 1 h. Sections were counterstained with DAPI and mounted with SlowFade Gold (Invitrogen). Cell counts were done in three control–KO littermate-matched pairs in a blinded manner.
6
ATRX Chromatin Immunoprecipitation in U-2 OS
7
ATRX Immunoprecipitation from U-2 OS Cells
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U-2 OSATRX nuclear extracts were prepared using Cellytic Nuclear Extraction kit (Sigma) in the absence of detergent and equilibrated into IP buffer (20 mM HEPES pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA+protease inhibitor cocktail). Immunoprecipitations were performed overnight at 4 °C using 10 μg anti-RAD50 antibody (Abcam ab89) or 10 μg anti-MRE11 antibody (Abcam ab214) with protein G dynabeads. Beads were washed four times in IP buffer and bound proteins eluted into SDS loading buffer (Laemmli) by heating at 90 °C for 5 min. Western blotting was performed using anti-ATRX antibody (Santa Cruz sc-15408).
8
Chromatin Remodeling Protein Analysis
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H3 general (ab1791, Abcam), H3.3 (09-838, Millipore), H3.1/2 (ABE154, Millipore), H4 (rabbit antiserum), H3K9me3 (ab8898, Abcam), Hira (mouse monoclonal WC15 and WC119), DAXX (sc-7152, Santa Cruz Biotechnology), ATRX (sc-15408, Santa Cruz Biotechnology), KAP1 (ab22553, Abcam; ab10483, Abcam), Tubulin (TUB2.1, Sigma), Lamin (ab26300, Abcam), normal rabbit IgG (12-370, Millipore).
9
Quantifying ATRX Protein Expression
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800 C3H/10T1/2 cells were seeded in a 96 well plate (glass bottom culture plates, MatTek, PBK96G-1.5-5-F) and keep cells in 37-degree for 24 h. Cells were fixed with 4% formaldehyde in PBS for 10 minutes at room temperature. Cells were permeabilized with 0.2% digitonin (EMD Millipore, 300410) in PBS (Corning, 21-040-CV) for 10 minutes at room temperature. Next, the cells were incubated in 3% BSA (in PBS, Sigma, #9418) for blocking for 1 h at room temperature followed by incubation in primary antibody (ATRX, Santa Cruz, sc-15408, 1:200) at 4-degree overnight, followed by secondary antibody (1:600, Invitrogen, A32754) incubation at room temperature for 1 h. Cells were washed three times with PBS for 5 minutes followed by DAPI staining (2 μg/ml in PBS, Sigma, D9564) for 5 minutes at room temperature. Mounting media (Vectashield, H-1000) was added immediately after DAPI staining. Cells were imaged using Widefield Microscope CellDiscoverer7 (CD7) automated widefield high-throughput system (Zeiss). Images were processed with ImageJ software (http://rsb.info.nih.gov/ij/ ). For the ATRX antibody, the ImageJ Brightness/Contrast was set as 30/112. For DAPI, the Brightness/Contrast was set as 37/123. All images were processed with the same parameter settings for each antibody.
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