Dylight488 conjugated donkey anti rabbit igg

2008/WT cells were seeded in 24-well plates on sterile glass coverslips and transfected with pcDNA3.1/Gβ1-myc-His expression vector (kindly provided by Prof. David Meiri, Dept. of Biology, Technion, Haifa, Israel) using linear polyethylenimine (PEI, MW 25,000) transfection reagent (Polysciences, Pennsylvania, USA) at a ratio of 3μg PEI : 1μg DNA. 20h after transfection, cells were incubated in the presence of either 20 nM EC0347 or 20nM EC1820 for 4h. Then, cells were washed with medium, fixed [4% formaldehyde in PBS, for 15min at room temperature (RT)] and permeabilized using 0.1% Triton X100 for 5 min. Blocking was performed with a blocking solution (20% skimmed milk in TBS) for 1hr at RT followed by co-incubation with primary anti-myc antibody (1:250, Abcam, Cambridge, UK) and anti-α-tubulin (1:500, Sigma-Aldrich, St. Louis, MO, USA) in 20% blocking solution for 1hr at RT. Coverslips were washed 3 times with PBS and co-incubated with Dylight 488-conjugated donkey anti-Rabbit IgG and Dylight 594-conjugated donkey anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA), for 1hr at RT. After three washes with PBS, coverslips were mounted onto glass slides using Fluoromount-G (Southern Biotechnology Associates) and examined using a confocal Zeiss LSM 710 microscope.

Custom Cep120, Ta3, Odf2, Cep164 antibodies were generated by Covance, Inc. Rabbits were immunized with insoluble His-tagged mouse Cep120 (660-E aa), Ta3 (1–430 aa), Odf2 (full-length), Cep164 (1–298 aa) protein fragments purified from bacteria. All these antibodies were used at a 1∶1000 dilution for both immunoblotting and immunostaining. Other antibodies used for immunofluorescence and Western blotting included Flag M2 mAb (1∶1000), acetylated tubulin (1∶2000), γ-tubulin (1∶2000) (Sigma), Arl13b (1∶1500) [24] (link), Calb1 (1∶200, cat #13176, Cell signaling), Pax6 (1∶50), and BrdU (1∶200) (DSHB, University of Iowa). Secondary antibodies Dylight 488-conjugated donkey anti rabbit IgG and Cy3-conjugated donkey anti mouse IgG were purchased from Jackson Immunoresearch, Inc.

A2780/SRSF3si2 cells were grown on poly-L-lysine-coated glass coverslip in the presence or absence of Doxy for 3 days before subjected for staining. The cells were fixed in ice-cold methanol for 10 min followed by air-dry. Afterwards, the cells were blocked in 5 % normal donkey serum (Jackson ImmunoResearch, West Grove, PA) for 1 h before they were incubated with γ-H2AX antibody (Cell signaling Technology, Cat # 9718S, 1:400 dilution) for 1 h and then with Dylight 488-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Cat # 711-485-152, 1:200 dilution) for 45 min. The cells were rinsed in 1xPBS for three times after each incubation step. Finally, the coverslips were mounted on glass slides with VECTASHIELD Mounting Medium containing 4′, 6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories, Burlingame, CA).