Mrfp rab7

Source of the antibodies are as follows: control mouse IgG, monoclonal anti-Rab7, rabbit polyclonal anti-LAMP2, and rabbit polyclonal anti-Stx17 (Sigma-Aldrich), monoclonal anti-dynein IC74 (EMD Millipore), monoclonal anti-dynactin p150^Glued (BD), rabbit polyclonal anti-DHC (Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-EEA1 (Cell Signaling Technology), and rabbit polyclonal anti-snapin (Synaptic Systems). mRFP-Rab7 (promoter: cytomegalovirus [CMV]; backbone: pmRFP-C3) was purchased from Addgene. Full-length snapin was cloned into the Eco RI–Kpn I sites of the pCMV-HA vector (Takara Bio Inc.) using standard PCR techniques. Site-directed mutagenesis to generate HA-snapin-L99K was performed using standard PCR techniques. GFP-LC3 was subcloned into pEGFP-C3 with CMV promoter. DIC-2C–mRFP (promoter: CMV; backbone: mRFP-N1) is a gift from K. Pfister (University of Virginia, Charlottesville, VA). These constructs were verified by DNA sequencing.

All primer sequences used in these experiments are listed in Supplementary Table 1. MT, 2xMT, CA, CA-MT, and CA(C258S) were generated by PCR amplification of pcDNA3.1(−)-Flag-RavZ and pcDNA3.1(−)-Flag-RavZ(C258S) vectors and inserted into the N3-GFP or C1-GFP vectors using each restriction enzyme set. CA(Δα3)-MT was amplified by overlap extension PCR with C1-CFP-CA-MT used as a template, and then was inserted into the C1-GFP vector using the restriction enzyme set. GFP-AKT1-PH, GFP-Lact-C2, mRFP-Rab5 and mRFP-Rab7 were provided by Addgene (Cambridge, MA, USA). We used previously described DNA constructs for mRFP-LC3B, and mRFP-GABA RAP in this study.