(a–o) All wing disc images shown are obtained from wandering third instar larvae. All transgenes are driven with dppblk-1-Gal4. Scale bars, 50 μm. Phospho-PDH-specific antibody is used to detect inactivation of PDH (a, b, e, f, green). Phospho PDHK, phospho JNK, and phospho Src-specific antibodies are used to detect activation of PDHK, JNK and Src respectively (c, d, g, h, green). Total PDHK protein expression detected using a α-PDHK1 antibody (i–o, green). The dppblk-1-Gal4, UAS Pvractcontrols are shown in the main Figure 5. (a–h) Src/JNK but not ERK/PI3K regulates activity of PDHK and PDH. Compared to Pvract control (see Figure 5f, green), knockdown of PDHK (a), hep (JNKK) (e), or Src42A (f) reduces p-PDH expression. Co-activation of ERK and PI3K pathways is not sufficient to inactive PDH (b, green) or to activate PDHK(c, green). While co-activation of ERK, PI3K and JNK can induce mild p-PDHK expression (d, green). Co-activation of ERK and PI3K pathways is not sufficient to active Src (g, green) or JNK (h, green). (i–q) Both ERK and PI3K pathways are required for PDHK translation. Knockdown of Src42A (i), or block of JNK (j) does not alter PDHK protein expression (green). Single expression of PI3Kact (k) or hRafact(l) is not sufficient to increase PDHK protein (green) amount. Reduction of Akt (m), Dsor1 (n), or elF4E (o) suppresses PDHK expression (green). (p–q) Real-time RT-PCR analysis of LDH and PDHK transcripts. The data were normalized with respect to the expression of RpL10 transcripts. Each experiment is done in triplicate. Compared with control, LDH level significantly increases in Pvract (p=0.0001, p) or hRafact+PI3Kact(p=0.0005, q) background, whereas PDHK level does not (p=0.6577, p; p=0.1941, q).