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3 protocols using mouse anti histone h2ax pser139

1

Immunofluorescence Staining of HeLa and Flp-In T-REx 293 Cells

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HeLa Kyoto cells or Flp-In T-REx 293 cells were fixed and processed for immunofluorescence as described45 (link). In brief, cells were fixed in 4% paraformaldehyde for 10 min at the room temperature followed by ice-cold methanol for 1 min. The cells were then wash with 1 × phosphate-buffered saline (PBS) and permeabilized with 0.5% Triton X-100 for 5 min before antibody staining. The primary antibodies used were as follows: mouse anti-histone H2AX pSer139 (1:1,000, Millipore 05-636-1), mouse anti-α-tubulin (1:5,000, Sigma-Aldrich T9026), rabbit anti-GFP (1:5,000, Abcam ab290), mouse anti-cyclin A (1:200, Santa Cruz sc-56299), mouse anti-FLAG (1:1,000, Sigma-Aldrich A8592). Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 (1:1,000, Molecular Probes) were used for detection. DNA was stained with 4,6-diamidino-2-phenylindole. Images were acquired using Zeiss AXIO Imager M1 with a × 40 EC-Plan-Neofluor lens and Hamamatsu photonics camera under the control of Volocity software. Images were processed using Adobe Photoshop.
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2

Immunofluorescence Imaging of Cell Markers

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HeLa Kyoto cells or Flp-In T-REx 293 cells were fixed and processed for immunofluorescence as described45 (link). In brief, cells were fixed in 4% paraformaldehyde for 10 min at the room temperature followed by ice-cold methanol for 1 min. The cells were then wash with 1xPBS and permeabilised with 0.5% Triton X-100 for 5 min before antibody staining. The primary antibodies used were: mouse anti-histone H2AX pSer139 (1:1000, Millipore 05-636-1), mouse anti-α-tubulin (1:5000, Sigma-Aldrich T9026), rabbit anti-GFP (1:5000, Abcam ab290), mouse anti-cyclin A (1:200, Santa Cruz sc-56299), mouse anti-FLAG (1:1000, Sigma-Aldrich A8592). Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 (1:1000, Molecular Probes) were used for detection. DNA was stained with DAPI. Images were acquired using Zeiss AXIO Imager M1 with a 40x EC-Plan-Neofluor lens and Hamamatsu photonics camera under the control of Volocity software. Images were processed using Adobe Photoshop.
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3

Detailed Western Blotting Protocol Using Phos-tag

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Western blotting was carried out as described23 (link). Phos-tag SDS-PAGE gels were made from 7% acrylamide (37.5:1 crosslinking ratio), 70 μM Phos-tag reagent (Wako Pure Chemical Industries) and 140 μM MnCl2, and were processed according to the manufacturer’s manual. Proteins were detected using the following primary antibodies: mouse anti-GFP (1:1000, Roche 11814460001), mouse anti-PLK1 (1:2000, Santa Cruz sc-17783), mouse anti-histone H3 pSer10 (1:5000, Abcam 14955), mouse anti-α-tubulin (1:5000, Sigma-Aldrich T9026), rabbit anti-phospho-(Ser) CDK substrate (1:1000, Cell Signaling 2324), rabbit anti-histone H3 (1:2000, Abcam ab1791), mouse anti-FLAG HRP (1:1000, Sigma-Aldrich A8592), mouse anti-MUS81 (1:1000, Santa Cruz sc-47692), mouse anti-BLM (1:500, Santa Cruz sc-70426), rabbit anti-KAP-1 pSer842 (1:1000, Abcam ab70369), rabbit anti-CHK1 pSer345 (1:1000, Cell Signaling 2341), mouse anti-histone H2A.X pSer139 (1:1000, Millipore 05-636-1), rabbit anti-GEN1 (1:100, raised against GEN1890-908)19 . Uncropped blots are show in Supplementary Fig. 5.
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