Licor fc odyssey

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.

Cells were harvested and lysed in RIPA buffer containing Halt protease inhibitor cocktail (Thermo Fisher Scientific) and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The samples were then sonicated, centrifuged for 30 min at 4 °C and the supernatants were collected. The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher, #23227). SDS-PAGE was performed approximately with 30 µg of protein, and proteins were transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk prepared in TBST buffer. After overnight incubation with primary antibodies at optimized dilutions (Table S2), membranes were washed and incubated with HRP conjugated secondary antibodies. Alternatively, blots were incubated with primary antibodies to proteins of interest together with GAPDH. Protein bands were detected using ECL Start Western Blotting Detection Reagent (Thermo Fisher, #32106) and imaged on an LICOR Fc Odyssey. (LI-COR Biotechnology Lincoln, Nebraska) and the band intensities were quantified using ImageJ (NIH).