Mouse anti myc 9b11

A 25–50 µg of TX-100 soluble material was separated by 3–15% gradient or 12% SDS–PAGE prior immunoblotting and chemiluminescence detection (Pierce). Antibodies used in this study were anti-RUY4 raised in rabbit against peptides 375–391 and 454–470 of mouse RUFY4. Mouse Anti-Myc (9B11, Cell Signaling), mouse Anti-Flag (M2, Sigma), rat anti-LAMP1 (134B, Biolegend), mouse anti-AIF (E-1, Santa Cruz), mouse anti-SDHA (2E3GC12, Abcam), mouse anti-LC3 (2G6, NanoTools), mouse anti-ß-actin (AC-15, Sigma). Secondary antibodies were from Jackson Immunoresearch, Molecular Probes (USA) and from Cell Signaling Technology. For immunofluorescence, cells on coverslips were fixed with 3.5% paraformaldehyde and permeabilized with 0.1% Triton X-100. Images were taken by a Zeiss LSM780 or Leica SP5 confocal microscope using 63× or 40× objective. Processing and quantification was performed using Fiji software [37 (link)]. Co-localization was quantified using JACoP plugin [38 (link)]. Statistical analysis was performed using Graphpad Prism. For two sets of values, we used t-tests, for multiple sets of values one-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. MitoTracker DeepRed and MitoTracker Green staining was performed according to the manufacturer's instructions (Thermofhisher) and detected by flow cytometry.

Immunohistochemistry was performed as described previously^{57 (link)}. The following antibodies were used: mouse anti-myc (9B11, 1:500; Cell Signalling Technology), mouse anti-pdf (C7, 1:1000; Developmental Studies Hybridoma Bank) and anti-mouse Alexa Fluor 568 (1:400; Thermo Fisher Scientific). The specimens were mounted using Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Images were captured using an FV3000 (Olympus) confocal microscope and processed using Imaris software (Bitplane). For quantification of the localisation pattern of myc-DapmaCRYA-D and myc-Drome-CRY in cell bodies of l-LN_v or s-LN_v, one- to four-day-old adult male flies were entrained to a 12-h:12-h light-dark cycle (LD; light: 200 lux) at 25 °C for 3 d. A total of 10–13 brains were immunostained and imaged at each time point (ZT 1, 5, 9, 13, 17 and 21). The areas of nucleus and cytoplasm were selected using freehand selection in Fiji, an open-source image analysis software^{58 (link)}. Selection of the regions of interests (ROIs) was performed by experimenters who were blind with respect to the genotype. The mean fluorescence intensities of the ROIs were then calculated.