Anti flag

For confocal analyses, we dissected at least six to ten animals. For fixation larvae were opened at the dorsal midline and stretched out with four needles, fixed in PBS 4% PFA for 20 min at room temperature and then washed with three quick buffer exchanges and three 20 min long washing steps. Tissues were blocked for immunohistochemistry with 10% goat serum for 1 h as described^{43 (link)}. The following antibodies were used: Anti-V5 (1:500, Invitrogen, R96025); anti-Flag (1:1000; Novus biologicals, NBP1-06712); anti-HA (1:1000; Covance, MMS-101P 901503); anti-HRP-DyLight^TM649 (1:500; Dianova, 123-165-021); anti-GFP (1:1000; Invitrogen, A6455); anti dsRed (1:1000, Clontech Labs 3P 632496), anti-Innexin2 (1:100^{67 (link)}) anti-Futsch (1:5, mAb 22C10^{71 (link),72 (link)}), conjugated secondary antibodies (all 1:1000, anti-mouse 488, A10680; anti-mouse 568, A11031; anti-rabbit 488, A11008; anti-rabbit 568, A11011; anti guinea pig 647, 1903515; anti-rat 647, A21247; all Invitrogen). All specimens were analyzed using a Zeiss 710 or 880 LSM; orthogonal sections were taken using the Zeiss LSM imaging software.

The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The protein-containing PVDF membranes were then probed with following primary antibodies: Anti- PKM1 (Cell Signaling Technology,7067S), Anti-PKM2 (Cell Signaling Technology, 4053S) to identify the level of PKM isoform, Anti- BORIS (Millipore ABE631), AntiDNMT3B (Abcam, ab13604), anti-flag (Novus Biologicals, NBP1-06712SS) and Anti GAPDH (Cell Signaling Technology, 5174S) were used as loading controls for protein assays. After 2 h incubation with primary antibody at room temperature (RT), membranes were washed with 1X tris-buffered saline and Tween-20 (TBST) then again incubated with secondary antibodies for 45 min at RT. The probed PVDF membranes were washed, and the bands were visualized using an Odyssey membrane Scanning system (Li-Cor Biosciences, Bad Homburg, Germany).