Lr cloning

pDest40-APEX2-V5 and pLEX_307-APEX2-V5; pDest40-RhTRIM5-APEX2-V5 and pLEX_307- RhTRIM5-APEX2-V5; pDest40-HuTRIM5-APEX2-V5 and pLEX_307-HuTRIM5-APEX2-V5; LIR2 mutant pDest40-GFP-RhTRIM5 were generated using Gateway recombination cloning. First, they were PCR amplified from available cDNA clones and recombined into pDONR221 using the BP reaction (Life Technologies, 11789–020) prior to being recombined into expression plasmids by LR cloning (Life Technologies, 11791–020). Plasmid constructs were verified by DNA sequencing. The AP1 luciferase reporter plasmid was a gift from Alexander Dent (Addgene plasmid #40342; 3XAP1pGL3), the NF-κB luciferase reporter was purchased from Promega (#E8491) and the Renilla luciferase plasmid (pRL-SV40, Addgene plasmid #27163) was a gift from Ron Prywes. All other plasmids have been previously published [16 (link),32 (link)]. All siRNA smart pools were from Dharmacon. siRNA was delivered to cells using Lipofectamine RNAiMAX (ThermoFisher, 13778150). Plasmid transfections were performed using Lipofectamine 2000 (ThermoFisher, 11668019) or Calcium Phosphate (Promega, E1200). Samples were prepared for analysis the day after DNA transfection. For siRNA experiments, cells were harvested 48–72h after siRNA transfection.

Wild-type ptc enhancers were amplified by PCR (Roche Expand High Fidelity PCR System) from BAC DNA (CH322-170A-12 or CH322-188E13). PCR primers are provided in Figure 2—source data 1. Enhancer constructs were sub-cloned into the pENTR/D-TOPO plasmid (Life Technologies) by TOPO cloning. Enhancers tested with the hspmin promoter, taken from the D. melanogaster Hsp70 gene, were subsequently cloned into the pHPdesteGFP transgenesis vector via LR Cloning (Life Technologies). Enhancers tested with the endogenous ptc promoter were cloned by traditional methods into the pStinger transgenesis vector (Barolo et al., 2000 ). Targeted GBS mutations were created by overlap-extension PCR (Swanson et al., 2010 (link)). Promoter analysis described in Figure 5 and Figure 5—figure supplement 3 was done by replacing the minimal hsp70 promoter contained in pHPdeste with the designated ptc promoter region (see Figure 2—source data 1 for sequences and restriction sites used). All enhancers and promoters were screened by restriction digest and sequencing.