Anti plk1 f 8

Antibodies used in this study include anti-phospho-210 PLK1 (BD 558400), anti-γ-tubulin (Thermo MA1-20248, clone GTU-88), anti-α-tubulin (MAB1864 Millipore), anti-anillin (polyclonal rabbit, Kim and Burkard, unpublished), anti-Plk1 (F-8, Santa Cruz Biotechnology sc-17783), anti-β-actin (AC-15, ab6276), anti-flag (M2) HRP (Sigma A8592), anti-RhoA (SC418 Santa Cruz), anti-pericentrin (ab44448 Abcam), anti-ACA (HCT0100 Immunovision), and anti-mouse HRP (Jackson Immunoresearch Laboratories Inc. #115-035-003). Immunoprecipitation of flag constructs was performed using anti-flag M2 affinity gel (Sigma A2220). For immunofluorescence, Alexa-flour antibodies were used (Invitrogen). Mitotic index was determined through Hoechst 33258 staining and microscopy. Crystal violet stain is composed of crystal violet (Sigma C-0775) with buffered formalin (Sigma HT-50-1-128)

His-PBD_326-603 was incubated with different compounds for the indicated period of time at room temperature in a buffer composed of 10 mM NaH₂PO₄ pH 8.0, 50 mM NaCl, 1 mM EDTA, 10% glycerol and 0.01% NP-40. Laemmli 2x buffer was added to stop the reaction and samples were boiled for 5 minutes, then subjected to SDS-PAGE on 12% acrylamide gels and then blotted on nitrocellulose membranes. Membranes were blocked with TBS-Tween 0.1% containing 5% of dry milk powder, then incubated with anti-Plk1 (F-8; Santa Cruz Biotechnologies Inc.) or with a 6x-His epitope tag antibody (Invitrogen; gift from Gregory Emery). HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories inc.) was used and TBS-Tween 0.1% was used for the washes before and after the secondary antibody. For the Coomassie Blue gel staining, gels were first fixed for 5 minutes in a fixing solution containing 40% ethanol and 10% glacial acetic acid, stained in a solution containing 50% methanol, 10% glacial acetic acid and 0.1% Coomassie Blue, and destained with a solution of 12% ethanol and 7% glacial acetic acid. Gels were imaged using a ChemiDoc MP system (BioRad).