Mouse anti cyclin a

Protein concentration was determined with the Bio-Rad DC protein assay. Protein samples were resolved on 10% or 13% polyacrylamide gels in an SDS running buffer. After incubation with the primary antibody and the corresponding horseradish peroxidase (HRP)–coupled secondary antibody, protein signals were detected by enhanced chemiluminescence (ECL) with a digital image analyzer ImageQuant LAS4000 from GE Healthcare, Chicago, IL, USA or IMAGEQUANT 800. The following antibodies were used: rabbit anti–p27^Kip1–C19 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti–p27^Kip1–horseradish peroxidase–coupled antibodies (clone 57, BD Biosciences, Franklin Lakes, NJ, USA), mouse anti–pY88–p27^Kip1 [14 (link)], rat anti–EpoR (GM1201, clone: BCO–3H2), rat anti–EpoR (GM1204) [22 (link)], mouse anti–Jak2 (clone 691R5, Invitrogen, Carlsbad, CA, USA), rabbit anti–pY1007/1008–Jak2 (Millipore, Burlington, MA, USA), rabbit anti–pY396 Lyn (Labome/Epitomics, Burlingame, CA, USA), rabbit anti–pY694 STAT5 (Cell Signaling, Danvers, MA, USA), mouse anti–Cyclin A (clone E67.1, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti–Cyclin E (clone HE12, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti–Cyclin A polyclonal antibody T310 [23 (link)], mouse anti–GAPDH (clone 6C5, Millipore, Burlington, MA, USA), and mouse monoclonal anti–PSTAIR [24 (link)].

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.