Monoclonal anti tubulin alpha

For western blots using whole animals, either roughly equal numbers of synchronized Day 1 adult animals were manually transferred into the lysis buffer (150 mM NaCl, 1 mM EDTA, 0.25% SDS, 1.0% NP-40, 50 mM Tris-HCl [pH7.4], Roche complete protease inhibitors and phosSTOP phosphatase inhibitors) supplemented with the 4 x SDS loading buffer and immediately frozen at −80°C, or animals were collected for extracting the total proteins via sonication and quantifying the protein concentrations via Bradford assays. For western blots using dissected tissues, approximately equal amount of biomass in each sample was collected into the same lysis buffer supplemented with the 4 x SDS loading buffer. Samples were boiled for 10 minutes before resolving on precast SDS-PAGE gels (GenScript). Bands of interests were quantified using the ImageJ software and normalized to the intensities of the internal control, which is either α-tubulin or β-actin. Antibodies used in western blots include monoclonal anti-FLAG (Sigma, 1804), monoclonal anti-Tubulin Alpha (Sigma, T6074), anti-Actin (CST, 4967), anti-Phospho-AMPKα (CST, 2535S) and anti-RPS-0, RPS-3, RPL-5 and RPL-25.2 antibodies (Liu et al., 2018 ).