Fig. 4
TBPH in neurons promotes synaptic growth through the regulation of Dlg levels. a qRT-PCR analysis of mRNAs immunoprecipitated by Flag-tagged TBPH (Elav-GAL4/UAS-TBPH/+;+/+, IP-TBPH) and its mutant variants TBPHF/L (Elav-GAL4/+;UAS-TBPH F/L/+, IP-TBPHF/L) in adult heads. The dlg enrichment folds was referred to rpl-11 (negative control); syntaxin has been used as the positive control. n = 3 (biological replicates). Individual data values are provided in the supplementary file data values. Individual data values are provided in Additional file 4. Individual Data Values.xls. b Western blot analysis of lane 1 (w1118), lane 2 (tbphΔ23/tbphΔ23), lane 3 (tbphΔ142/tbphΔ142), and lane 4 (tbphΔ23,Elav-GAL4/tbphΔ23,UAS-TBPH). Adult brains, 1 day old, were probed with anti-Dlg antibody and anti-alpha-tubulin as loading control. Quantification of normalized amounts was reported below each lane. n = 3 (biological replicates). Individual data values are provided in the supplementary file data values. c qRT-PCR analysis of DLG. mRNA from adult heads of the Ctrl (w1118) and the two null alleles of TBPH (tbphΔ23/tbphΔ23 and tbphΔ142/tbphΔ142) were retrotranscribed with oligo-dT and random hexamers for exons 1–4 and introns 1–4, respectively; DLG transcripts were normalized on Sdha reference gene, and folds were calculated versus wildtype control. n = 2 (biological replicates; real-time reactions performed in triplicate for each biological replicate). Individual data values are provided in the supplementary file individual data values. **p < 0.01, ***p < 0.001, and ****p < 0.0001 calculated by one-way ANOVA, error bars SEM. d Number of peristaltic waves of Ctrl (w1118), Δtb-GFP (tbphΔ23/tbphΔ23;nSyb-GAL4/UAS-GFP), and Δtb-Dlg (tbphΔ23,UAS-Dlg/tbphΔ23;nSyb-GAL4/). n = 20. e Confocal images of third instar NMJ terminals in muscle 6/7 second segment stained with anti-HRP (in green) in Ctrl, Δtb-GFP, and Δtb-Dlg. f Quantification of branch number. n = 15. g Confocal images of third instar NMJ terminals in muscle 6/7 second segment stained with anti-HRP (in green) and anti-GluRIIA (in red) in Ctrl, Δtb-GFP, and Δtb-Dlg. h Quantification of GluRIIA intensity normalized on Ctrl. n > 200 boutons. **p < 0.01 and ***p < 0.001 calculated by one-way ANOVA, error bars SEM. Scale bar 10 μm (in e) and 5 μm (in g)