Fly Head Protein Extraction and Western Blot

Fly heads were collected and mashed in a sample lysis buffer. The samples with SDS sample buffer were boiled and run on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes. Primary antibodies for PO₄-CREBB (1:1000), pan-CREBB (1:5000) (Fropf et al., 2013 (link)), CREBA (1:5000) (Andrew et al., 1997 (link)) (Developmental Studies Hybridoma Bank), HA-tag (1:2000) (BioLegend), His-tag (1:2000) (Clontech) and actin (1:10,000) (Abcam) were used and detected by HRP conjugated secondary antibody (1:10,000) (Jackson ImmunoResearch). For detecting the ATG2-CREBB protein (Fropf et al., 2013 (link)), 50 adult brains were dissected and collected in 20 μL of the sample lysis buffer and placed at −80°C for overnight. After vortexing for one minute, samples were mixed with SDS sample buffer and boiled before loading into an SDS-PAGE gel and transferred to nitrocellulose. Primary antibodies for ATG2-CREBB (1:1,000) (Fropf et al., 2013 (link)) was used and detected by HRP conjugated secondary antibody (1:10,000) (Jackson ImmunoResearch). An ECL reagent kit (ThermoFisher) was used for Western blot detection.