Irdye 800 labeled anti mouse igg

The isolation of fly mitochondria was performed as previously described,^{56 (link)} except that 20 eyes were gently homogenized in 1 ml of mitochondrial isolation buffer (250 mM sucrose, 10 mM Tris (pH 7.4), and 0.15 mM MgCl₂). The mitochondrial (pellet) and cytosolic (supernatant) fractions were solubilized in a 50 μl SDS sample buffer and then subjected to western blotting.
In order to detect HA-tagged PINK1 and Flag-tagged Parkin on western blots, the samples were homogenized in an SDS sample buffer, fractionated by SDS-PAGE, and transferred to Immobilon-FL membranes (Millipore, Billerica, MA, USA). The blots were then incubated with mouse anti-HA (Roche, Indianapolis, IN, USA) or mouse anti-Flag (Sigma) primary antibodies or control antibodies, which included mouse anti-Rh1 (Developmental Studies Hybridoma Bank, Ames, IA, USA), rabbit anti-CoIV (Abcam, Cambridge, MA, USA), and rat-anti-TOM20. The blots were subsequently incubated with IRDye 800-labeled anti-mouse IgG and IRDye 680-labeled anti-rabbit or rat IgG (LI-COR Biosciences, Lincoln, NE, USA). The signals were then detected using an Odyssey Infrared Imaging System (LI-COR Biosciences).

Mobility shift of phosphorylated dMyc protein was detected by phosphate affinity SDS-PAGE using acrylamide-pendant phos-tag (Phos-tag AAL-107). Briefly, 50–100 μM phos-tag acrylamide and 100–200 μM MnCl₂ were added to normal 6% polyacrylamide gel. After electrophoresis, the gel was washed with transfer buffer containing 1 mM EDTA for 10 min with gentle agitation, and then with transfer buffer without EDTA for 10 min. Proteins were transferred to Immobilon-FL transfer membranes (Millipore). For western blotting, pupae were homogenized in SDS sample buffer with a pellet pestle (Kimble-Kontes), and the proteins were fractionated using SDS-PAGE. The proteins were transferred to Immobilon-FL transfer membranes in Tris-glycine buffer. The blots were probed with mouse anti-HA 1:500 (Cell Signaling), mouse anti-dMyc (1:50), and rabbit anti-α tubulin 1:15000 (Sigma), and subsequently with IRDye 800-labeled anti-mouse IgG and IRDye 680-labeled anti-rabbit IgG (Licor). Signals were detected using an Odyssey infrared imaging system.