Anti c myc

All chemicals were of an analytical grade and, unless otherwise noted, from Sigma-Chemical or Fisher Scientific. [γ-32P]ATP (specific activity 3000 Ci/mmol) was from PerkinElmer. The MCOLN1 peptides (RRKCGRDPSEEHSLLVN, RRKCGRDPAEEHALLVN, RRKCGRDPAEEHSLLVN, RRKCGRDPSEEHALLVN) were synthesized by the UNC High-Throughput Peptide Synthesis and Array Facility. MHY1485 was synthesized and the compound identity was verified by LC–MS and NMR, in-house at North Carolina Central University, as previously described [35 (link)]. The antibodies used were as follows: anti-LC3 [1:1000 (in milk); MBL International], anti-c-Myc [1:40 dilution for 1 mg/ml lysate for immunoprecipitation (IP); Developmental Studies Hybridoma Bank, University of Iowa], anti-α-tubulin (clone B-5-1-2; Sigma–Aldrich) and anti-GFP (A11122 for IP and A6455 for Western; Molecular Probes). pEGFPC2-MCOLN1 [full-length, wild-type (WT), human and cloned into the EcoRI and SalI sites] was a generous gift from R. Puertollano (National Institute of Health, Bethesda, Maryland) and is as previously described [36 (link)]. The myc-TOR, kinase-dead (KD) myc-TOR and RFP-LC3 plasmids were from Addgene.