Irdye 800rd

200 adult worms from each strain were transferred to 30 μl of sample buffer (80.0 mM Tris, pH 6.8, 2.0% SDS, 10.0% glycerol, 0.0006% bromophenol blue, and 5.0% β-mercaptoethanol), subjected to the freeze-thaw treatment three times in liquid nitrogen and water bath and then boiled in a 95°C-heating block for 5 min. The samples were loaded on 10% SDS–PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane and subjected to Western blot analysis using anti-PLK-1 (1:3,000 dilution, gift from Monica Gotta laboratory, Tavernier et al [2015] (link)) and anti-GFP (1:3,000 dilution, ab6556; Abcam). Primary antibodies were detected using IRDye 680RD goat anti-rabbit (1:5,000 dilution; ab21677; Abcam) and then probed for α-tubulin using the monoclonal DM1α antibody (1:3,000 dilution; ab7291; Abcam) and IRDye 800RD goat anti-mouse (1:5,000 dilution; ab21677; Abcam) to examine the expression levels of α-tubulin as a loading control. Antibody signals were detected using the Azure Biosystems c600 and Li-COR Fc Odyssey imaging system. The pixel intensity of protein bands was then quantified with Image J (http://rsbweb.nih.gov/ij/) and normalized the signal to signal of α-tubulin.

Protein samples were separated on SDS-PAGE gels and then electroblotted onto nitrocellulose membranes. The nitrocellulose membranes were blocked for 1 h in 5% non-fat milk in TBST (10 mM Tris, pH 7.5, 200 mM NaCl, and 0.2% Tween 20) followed by incubation with primary antibodies: mouse anti-GFP antibody (Cat. 66002-1-Ig, Proteintech, Rosemont, IL, United States), rabbit anti-GFP antibody (Cat. ab290, abcam, Cambridge, United Kingdom), mouse anti-FLAG antibody (Cat. F3165, Sigma), rabbit anti-FLAG antibody (Cat. 20543-1-AP, Proteintech), rabbit anti-Myc antibody (Cat. 16286-1-AP, Proteintech), mouse anti-β-Actin antibody (Cat. 66009-1-Ig, Proteintech), rabbit anti-CDK2 antibody (Cat. 10122-1-AP, Proteintech), or rabbit anti-pCDK2 antibody (Cat. ab194868, abcam). Two secondary antibodies were used: goat anti-mouse antibody (Cat. ab216776, IRDye 680RD, abcam) and goat anti-rabbit antibody (Cat. ab216773, IRDye 800RD, abcam). Signals were captured by an Amersham Typhoon (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and analyzed by ImageQuant TL (GE Healthcare).