Computational protocol: Different effects of Atg2 and Atg18 mutations on Atg8a and Atg9 trafficking during starvation in Drosophila☆

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Protocol publication

[…] Western blots were carried out as described previously . Dissected larval carcasses were processed for LysoTracker Red (Invitrogen) or immunostaining as before, using rabbit anti-Ref(2)P (1:2000), rat anti-Atg8a (1:300), rat anti-Atg9 (1:300), rat anti-FIP200 (1:300) and rat anti-mCherry (1:300) primary antibodies . Preparations were photographed on an Axioimager M2 with Apotome2 (Zeiss), and original unmodified images were processed for statistical evaluation using ImageJ (NIH) and SPSS Statistics (IBM), as described . Colocalizations were calculated either by the colocalization tool in ImageJ to obtain Mander’s colocalization coefficients for Atg8a-Ref(2)P, or by manual counting for Atg9-Ref(2)P. Immunogold labeling for ubiquitin was done as described . Anti-Atg9 (1:30) and anti-Ref(2)P (1:60) double immunogold labeling was carried out on adult brains embedded into LR White resin (Sigma) to increase antigenicity, using secondary antibodies anti-rat conjugated to 10 nm gold (1:20, Sigma) and anti-rabbit conjugated to 18 nm gold particles (1:60, Jackson Immunoresearch). […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Drosophila melanogaster, Saccharomyces cerevisiae