Mouse anti plk1 f 8

Cell lysates was analyzed by SDS-PAGE, and transferred to nitrocellulose membranes by tank electrotransfer. Membranes were blocked using either 5% skim milk in Tris-Buffered-Saline (TBS), or with Odyssey blocking buffer (LI-COR BioSciences) for 1 hr at RT. Primary antibody probing was done either in 1% skim milk in TBS-T (TBS with 0.1% Tween-20), or in Odyssey blocking buffer, for 1-2 hr at RT, or O/N at 4°C. Primary antibodies used are Mouse-anti-FLAG M2 (1:1000; Sigma), Rabbit-anti-FLAG (1:1000; Sigma), Rat-anti-HA 3F10 (1:500; Roche), Mouse-anti-β-actin (1:10,000; Sigma), Rabbit-anti-β-Actin (1:1000, Cell Signaling Technology), Rabbit-anti-phospho-tyrosine P-Y-1000 (1:2000; Cell Signaling Technology), Mouse-anti-Plk1 F-8 (1:100; Santa Cruz Biotechnology, Inc) and Rabbit-anti-Plk1 (1:100; Santa Cruz Biotechnology, Inc). After primary antibody incubation, the membranes were washed 3x with TBS-T, followed by staining with secondary antibody in Odyssey blocking buffer for 1 hr at RT. Secondary antibodies used were Goat-anti-Mouse, Goat-anti-Rabbit, or Goat-anti-Rat, all from LI-COR, all 1:10,000, and all labeled with either IRDye 680 or IRDye 800. After secondary antibody staining, the membranes were washed again 3x with TBS-T, and imaged with an Odyssey CLx scanner (LI-COR BioSciences). Image analysis was done using ImageStudio (LI-COR BioSciences).

Mouse anti-Plk4 antibody has been described previously (Cizmecioglu et al., 2010 (link)) and used at a final concentration of 1 µg/ml. Mouse anti-Flag M2 (F3165), mouse anti-α-tubulin (T5168) and mouse anti-γ-tubulin (T6557) were from Sigma. Mouse anti-Myc (9E10), mouse anti-Plk1 (F-8), and mouse anti-cyclin E (HE12) were obtained from Santa Cruz Biotechnology. Mouse anti-actin (JLA20) was from Calbiochem, mouse anti-His from Qiagen, rabbit anti-GFP (NB600-303) from Novus, rabbit anti-CP110 (A301-343A) and rabbit anti-STIL (A302-442A) for western blotting from Bethyl, rabbit anti-STIL (ab89314) for immunofluorescence and rabbit anti-pericentrin (ab4448) from Abcam. Rabbit anti-cyclin B1 has been described previously (Hoffmann et al., 1993 (link)). Secondary antibodies for western blotting were peroxidase-conjugated donkey anti-rabbit (Jackson Laboratories) and goat anti-mouse (Novus). Secondary antibodies for immunofluorescence were goat anti-mouse IgG and goat anti-rabbit IgG coupled to Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes).