Quantifying Phosphorylated PMK-1 in C. elegans

C. elegans were prepared as described above to ensure that stage-matched, hermaphrodite animals at the young L4 larval stage were studied in each condition. Protein lysates were prepared as previously described [17 (link)] and probed with a 1:1000 dilution of an antibody that recognizes the doubly-phosphorylated TGY motif of PMK-1 (Promega Corporation). Monoclonal anti-α-tubulin antibody was used at a dilution of 1:1,000 (Sigma-Aldrich). A polyclonal antibody against the total PMK-1 protein was raised using the peptide DFQKNVAFADEEEDEEKMES (PMK-1 amino acids 358 to 377) in a rabbit (Thermo Scientific Pierce Custom Antibody Services) and used at a dilution of 1:1000. We confirmed that the total PMK-1 antibody detects total, but not active (phosphorylated) PMK-1 (Fig 5C). Horseradish peroxidase (HRP)-conjugated anti-rabbit (Cell Signaling Technology) and anti-mouse IgG secondary antibodies (Abcam) were diluted 1:10,000 and used to detect the primary antibodies following the addition of ECL reagents (Thermo Fisher Scientific, Inc.), which were visualized using a BioRad ChemiDoc MP Imaging System. The band intensities were quantified using BioRad Image Lab software version 5.2.1, and the ratio of active phosphorylated PMK-1 to total PMK-1 was calculated with all samples normalized to the ratio of wild-type control animals.