Pac5.1 v5 his

The copper-inducible pMT/V5-His plasmid (Invitrogen, Carlsbad, CA, USA) was utilized as a backbone vector for generating reporter constructs. For generation of pMT-Luciferase renilla (pMT-Renilla), the coding sequence of renilla luciferase was subcloned from a pRL-SV40 vector (Promega, Madison, WI, USA) into HindIII/XbaI sites of pMT/V5-His. For the pMT-Luciferase firefly reporter construct (pMT-Firefly), the firefly luciferase coding sequence was subcloned from pGL3 vector (Promega, Madison, WI, USA) into EcoRI/XbaI sites of pMT/V5-His. fatiga 3’UTR sequence was generated by PCR from cDNA prepared from Drosophila yellow white embryos and cloned into XbaI/ApaI restriction sites of the pMT-Firefly plasmid. The primers utilized were:
Forward (Fw): 5’-GCTCTAGACCCAAGCCGACAGCGCAGCT-3’;
Reverse (Rv): 5’-GCCATTGGGCCCCATCAGCTCAGGCTTTTGTTTA-3’.
Point mutations in miR-190 binding site at the fatiga 3’UTR were introduced by nested PCR with the following primers:
Fw: 5’-CTGTAAATCATGAAGTATGTATATTTATGCCCTCGCTACATATTGTATG-3’;
Rv: 5’-CATACAATATGTAGCGAGGGCATAAATATACATACTTCATGATTTACAG-3’.
The Luc-CG10011 3’UTR reporter and pAc-miR-12 were a gift from E. Izaurralde [44 (link)]. The pAc-miR-190 overexpression plasmid was kindly provided by M. Milán [104 (link)]. The pAc-5.1/V5-His (Invitrogen, Carlsbad, CA, USA) was used as a negative control.

S2 cells, wild-type S2R+ cells, and tsc2 KO S2R+ cells [44 (link), 45 (link)] were grown in Schneider’s Drosophila Media (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and penicillin–streptomycin. For insulin treatment, 25 µg/ml of insulin was treated for the indicated time lengths. For rapamycin (LC Laboratories) treatment, cells were incubated with 20 nM rapamycin for the indicated time lengths.
For RNAi experiments, PCR templates for dsRNA against CCT1 through CCT8 were prepared using primers designed by SnapDragon-dsRNA design (http://www.flyrnai.org/snapdragon). dsRNAs for CCT1-8 were generated by PCR using MEGAscript T7 (Ambion) and purified using MEGAClear (Ambion). Thirty micrograms of dsRNA was treated in S2R+ cells in six-well plates for 3 days using bathing method [46 (link)].
For immunoprecipitation between Myc-CCT4 and Rheb-V5 proteins, coding sequences of CCT4 and Rheb were cloned into pAc5.1-V5/His (Invitrogen) with N-terminal Myc tag with and without C-terminal stop codon, respectively. The cloned constructs were transfected in S2 cells using Effectene reagent (Qiagen).

All Abi mutants were generated using the Quikchange Site-directed mutagenesis Kit (Stratagene). Abi and EVH1 binding mutants were cloned into CB6-N-GFP and pAC-C-GFP (Ac5 promoter) modified from pAc5.1/V5-His (Invitrogen) for expression in mammalian and Drosophila cells, respectively. 293T and S2 cells were transfected with Abi expression vectors using calcium phosphate or Effectene (QIAGEN). Coimmunoprecipitations and pull-down assays using GST-EVH1-Ena were performed as described previously (Boëda et al., 2007 (link), Boëda et al., 2011 (link)). Far western analysis of overlapping 20-mer Human and Drosophila Abi peptide arrays were probed with GST or GST-tagged EVH1 domains of Ena, Evl, Mena, and VASP as described previously (Postigo et al., 2006 (link)).

Hrs, Golgin84, Golgin245 (lacking the GRIP domain), Rab7 and Gmap (amino acids 1-1356) were inserted into a modified version of pAc5.1-V5-His (Invitrogen) containing an N-terminal GFP cassette and a stop codon before the V5 tag. Cnx99A was tagged with myc at the C-terminus using the Gateway^® vector pAWM (Life Technologies). S2 cells were plated in 75 cm² flasks and transfected with 15 µg GFP-tagged construct and 15 µg carrier DNA using Fugene^HD (Roche) according to the manufacturer's instructions. Cells were incubated at 25°C for 48 h before scraping into ice-cold PBS, pelleting and resuspending in lysis buffer [20 mM Tris-HCl pH 7.4, 110 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5% (w/v) CHAPS plus protease inhibitors]. Cells were incubated for 30 min at 4°C then clarified by centrifugation and pre-cleared by incubation with protein G PLUS-agarose (Santa Cruz) for 30 min at 4°C. Lysates were collected and incubated with 200 µl monoclonal tissue culture supernatant or 20 µl polyclonal goat serum for 2 h at 4°C with rotation. protein G PLUS-agarose was added and the samples incubated for a further 2 h at 4°C with rotation. Beads were then washed in lysis buffer and isolated tagged proteins eluted with sample buffer. Samples were separated by SDS-PAGE, transferred to nitrocellulose and probed with mouse anti-GFP (Roche), rabbit anti-myc (Santa Cruz), or anti-Golgin245 antibodies.