Pxi western blot imager

Whole flies (5 flies per sample) were homogenized in Lysis Buffer (PBS 1X, Protease Inhibitors 1X, NuPAGE LDS Sample Buffer 1X, and DTT (Dithitreitol) 0.05M). For Triton fraction, 10 flies were homogenized in Triton Buffer (Triton X-100 1%, PBS 1X and Protease Inhibitor 1X). Samples were spun at 13000 × g for 5 min. at 4°C, then supernatant (Triton fraction) was transferred to a new vial containing 4X Buffer (NuPAGE LDS Sample Buffer 4X, and DTT 0.05M). Samples from whole flies or Triton fraction were separated by SDS-PAGE gels and proteins were transferred to Nitrocellulose membranes. Samples were collected and lysates were separated by SDS-PAGE using standard procedures. Membranes were probed with antisera against: anti-α-actin peroxidase conjugated 1:15000 (A3854, Sigma), mouse-anti-VDAC1 / Porin 1:10000 (ab14734, Abcam), rabbit-anti-dP62 1:2500 (3), anti-GABARAP (ab109364, Abcam), rabbit-anti-HA (3724, Cell Signaling) and mouse-anti-ubiquitin (P4D1) 1:1000 (3936, Cell Signaling). Anti-Rabbit or anti-Mouse Horseradish peroxidase conjugated antibodies were used for detection at 1:10000 dilution. Amersham ECL Prime Western Blotting Detection Reagent (GE life sciences) was used to visualize the presence of horseradish peroxidase, and the chemiluminescent signal was recorded using Syngene Pxi Western Blot Imager. Image analysis was done using ImageJ.