Peyfp c1

Genes for GABARAP, GABARAPL1 and GABARAPL2 were subcloned from GST-fusion plasmids (Addgene IDs 73948, 73945 and 73518) by PCR amplification into the XhoI and BamHI sites of peYFP-C1 or  peCFP-C1(Clontech), yielding peYFP-C1/GABARAP, peYFP-C1/GABARAPL1 and peCFP-C1/GABARAPL2.

Rat Kv1.3 and human Kv1.5 channels and the mitochondrial marker have been widely described by our laboratory (18 (link), 19 (link), 23 (link), 24 (link)). Rat Kv1.1, Kv1.2 and Kv1.4 channels in pGEM7 were obtained from M. M. Tamkun (Colorado State University, CO). Channel cDNAs were subcloned into pEYFP-C1 (Clontech). HA-Sar1 (H79G) was from R. Pepperkok (EMBL, Heidelberg, Germany). Kv1.3-ΔSx constructs were generated by inserting XhoI sites at the beginning and at the end of the Sx sequence in the Kv1.3 pEYFP-C1 plasmid. Mutations to introduce restriction sites were generated using the QuikChange multisite-directed mutagenesis kit (Agilent Technologies). YFP-Sx constructs were obtained by fusing the Sx segment to the C-terminus of pEYFP-C1 (Clontech). Forward and reverse oligonucleotide strands containing the Sx sequence were designed with EcoRI and BamHI sites at the beginning and at the end, respectively, to generate Sx segments. Oligonucleotides were annealed and cloned into the pEYFP-C1 plasmid. Primers designed to obtain the constructs are shown in Supplementary Tables 1 and 2. All constructs were verified by sequencing.

Complementary DNAs encoding murine Plin5 (NM_025874.3), Atgl (NM_001163689.1), or Cgi-58 (NM_026179.2) were cloned into the pcDNA4/HisMaxC expression vector (Invitrogen Life Technologies), as previously described (6 (link), 30 (link), 31 (link)). A pcDNA4/HisMax vector encoding Escherichia coli β-galactosidase (β-Gal) was provided by the manufacturer (Invitrogen Life Technologies). The detailed procedures for generation of pLVX-IRES-PURO (Clontech) constructs encoding recombinant proteins with an N-terminal FLAG tag as well as pEYFP-C1 (Takara Bio USA, Inc) constructs encoding target proteins with an N-terminal enhanced yellow fluorescent protein (EYFP) tag are described in the supplemental data section.

The βTrCP-EYFP was constructed by restriction enzyme cloning. Using p4489 Flag-βTrCP (Cat no. 10865, Addgene, Watertown, MA), βTrCP was amplified by PCR and ligated into pEYFP-C1 (TaKaRa Bio USA, Inc., Mountain View, CA) via the AgeI and NheI sites. The primers used in the PCR are as follows: forward 5′-TAATGCTAGCGCCACCATGGACTAC-3′ and reverse 5′-GAGCACCGGTCTTCTGGAGATGTAGGT-3′. The Kras-PDGFR biosensor was kindly provided by Dr. Jihye Seong (Korea Institute of Science and Technology, Republic of Korea). The Rac biosensor was a gift from Dr. Yingxiao Wang (University of California, San Diego, CA). JNKAR1EV-NLS was also constructed by restriction enzyme cloning. The DNA sequence of the Ypet-FHA1-EV linker-JNK substrate was excised from the hyBRET-JNK-EV construct (Cat no. 108656, Addgene) using EcoRI and NotI restriction enzymes. After 4048NLS, which contains the Ypet-ECFP FRET pair and the NLS, was excised using the same enzymes, the NLS-containing vector and insert containing the JNK-specific substrate were ligated. Dr. Michiyuki Matsuda (Kyoto University, Japan) kindly gifted the 4048NLS. Additionally, 3xAP1pGL3 (Cat no. 40302, Addgene) and pGL3-NFAT luciferase (Cat no. 17870, Addgene) were kind gifts from Alexander Dent and Jetty Crabtree, respectively.

Oligonucleotide primers designed to amplify chicken Ku70 cDNA were created to clone the coding sequence of Ku70 in-frame between XhoI and EcoRI sites of pEYFP-C1 (Takara Bio Inc., Shiga, Japan), based on the published Ku70 cDNA sequence of G. gallus domesticus (chicken) (DDBJ/EMBL/GenBank accession No. AB016529.1)^{5 (link)}. The sense primer (Ku70-Xho-F: 5′-CGCGGAACTCGAGCTatggccgactgggtgtcctattatc-3′) and antisense primer (Ku70-Eco-R: 5′-CCGTGAATTCttagcgcccactgaagtattcagtc-3′) incorporated XhoI and EcoR1 restriction enzyme sites, respectively. High-fidelity Platinum™ SuperFi™ DNA Polymerase (Thermo Fisher Scientific) was used for PCR amplification following the manufacturer's instructions. The PCR conditions included an initial denaturation step at 95 °C for 1 min, followed by 30 cycles of denaturation at 95 °C for 0.5 min, annealing at 60 °C for 0.5 min, and extension at 72 °C for 1 min. The PCR products were digested and ligated in-frame into the pEYFP-C1 vector using the DNA Ligation Kit, Mighty Mix (Takara Bio, Inc.), and the inserts were confirmed by sequencing.

pmApple-paxillin was from Mike Davidson (Florida State University). pEYFP-paxillin was generated by subcloning the sequence encoding human paxillin into the HindIII–XbaI sites of pEYFP-C1 (Takara Bio Inc.) that contained a modified multiple cloning site. pEGFP-tensin 1 was described previously52 (link). pEYFP-talin was generated by subcloning the sequence encoding human talin (from Richard Hynes) into the Not1-EcoR1 sites of pEYFP-NBC1 that contained a modified multiple cloning site. EGFP-lifeact was purchased from Ibidi. Plasmids were transfected into fibroblasts by electroporation using a Bio-Rad Gene Pulsar TM at 170 V, 960 μFd with external capacitance and a time constant of 17–22 μs in 0.4-cm gap cuvettes.