Anti dperk

Standard protocols were used to fix and stain wing discs for immunohistochemistry. TO-PRO-3 or DAPI was used for a DNA counterstain (Invitrogen, Carlsbad, CA), and tissues were mounted in Vectashield (Vector Labs, Burlingame, CA). The following antibodies were used: anti-dpERK (1:100; Sigma-Aldrich, St. Louis, MO), anti-phospho Drosophila Akt (Ser505) (1:25; Cell Signaling, Danvers, MA), anti-Sima (1:100; generated by PRF&L, Canadensis, PA), anti-phospho PDH (S293) (1:100; Abcam, Cambridge, MA), anti-PDHK1 (1:100; Abcam), anti-phospho PDHK1 (Tyr243) (1:200; Cell Signaling), anti-phospho JNK (Thr183/Tyr185) (1:500; Cell Signaling), and anti-phospho Src (Tyr418) (1:200; Invitrogen). Single- plane fluorescence images were obtained using a Zeiss LSM 700 confocal microscope with Zen 2009 acquisition software. Images and average intensity of images were processed and measured using ImageJ. At least 20 discs were used for each genotype per experiment. Three technical replications were done for each experiment.

Western blots were carried out under reducing conditions as previously described [26 (link)]. Lysate from five equatorial explants or from 1/5th of five embryos was loaded per lane. Antibodies used were anti-phospho-Smad2 (Cell Signaling Technology; 1 : 500), anti-dp-Erk (Sigma M8159; 1 : 5000) and anti-hemagglutinin (HA) high affinity rat monoclonal antibody 3F10 (Roche 1-867-423; 1 : 2000); anti-α-tubulin (DM1A Neomarkers, 1 : 5000) was used as the loading control. Phopho-Smad2 and dp-Erk western blots were repeated to ensure the reproducibility of the results.