Pmcherry c2 vector

A cDNA fragment encoding mouse Ulk1 was amplified from the genomes of wild-type MEFs with forward (5′-AAGGGATCCGAATTCATGGAGCCGGGCCGCGGCGGCG-3′) and reverse (5′-AGATGCATGCTCGAGTCAGGCATAGACACCACTCAGCAG-3′) primers. It was cloned into EGFP-pcDNA at EcoRI-XhoI sites, resulting in GFP-Ulk1-pcDNA. A cDNA for GFP-Ulk1 was amplified from GFP-Ulk1-pcDNA with forward (5′-ATTTCCGGTGAATTCATGGTGAGCAAGGGCGAG-3′) and reverse (5′-GGTAGAATTGGATCCTCAGGCATAGACACCACTCAGCAG-3′) primers and was cloned into pLVSIN-CMV-puro (TaKaRa), resulting in GFP-Ulk1-pLVSIN-CMV-puro. To generate pmCherry-p62, p62 was amplified by PCR and digested with EcoRI-XhoI. The PCR product was subcloned into the EcoRI-SalI site of the pmCherry-C2 vector (Clontech, Mountain View, CA).

To generate a construct encoding human Munc18-2 tagged at the N-terminus with a Myc-epitope, a two step PCR procedure was used. The primers Munc18NT-F1 and Munc18NT R1b were used in step 1 to amplify the Munc18-2 human cDNA clone (MGC clone 71251). The gel purified PCR product was then used as the template in a second PCR reaction and amplified with the primers Munc18NT-F2 and Munc18NT R2. The resultant PCR product was then cloned into pcDNA3.1Pac(-) using Eco R1 and Bam H1 to generate pCDNA3.1Pac(-)-Myc-Munc18-2. To generate a mCherry fusion, the Munc18-2 open reading frame was cut out of pcDNA3.1Pac(-)-Myc-Munc18-2 and inserted into the pmCherry-C2 vector (Clontech) using Eco R1 and Bam H1. Oligonucleotide sequences are provided in Table 1.