Anti vps26

Most general reagents used in this study were sourced from Sigma-Aldrich. The siRNA oligonucleotides were purchased from Dharmacon. Primary antibodies used in this study were as follows: anti-TBC1D5 [Santa Cruz, sc-376296, dilution 1:400 or 1:1000 for immunofluorescence (IF) microscopy or western blotting (WB), respectively], anti-VPS26 (Abcam, ab23892, 1:800 IF or 1:1000 WB), anti-VPS35 [Santa Cruz, sc-374372, 1:800 IF or 1:1000 WB, or from the Seaman lab (see Seaman, 2007 (link)), 1:300 for IF], anti-CIMPR (Abcam, ab2733, 1:400 IF or 1:1000 WB), anti-Lamp1 (Santa Cruz, sc-18821, 1:500 IF or 1:1000 WB), anti-Glut1 (Abcam, ab15309, 1:400 IF), anti-GM130 (BD Transduction labs 610822, 1:500 IF), anti-Fam21 (Millipore, ABT79, 1:400 IF or 1:1000 WB), anti-Aβ (Covance, SIG-39320, 1:1000 WB), anti-sAPPβ (IBL America, 10321, 1:800 WB), anti-Rab7a:GTP (NewEast Biosciences, 26923, 1:300 IF), anti-TGN46 (Seaman lab, see Seaman, 2007 (link), dilution 1:600 IF), anti-GFP (Seaman lab, see Seaman et al., 2009 (link), 1:1000 for immunoprecipitation), anti-Snx1 (BD Transduction labs, dilution 1:400 IF or 1:1000 WB) and anti-Tubulin (Sigma-Aldrich, dilution 1:1000 WB). Secondary fluorescently labelled antibodies were purchased from Invitrogen.

Vps35 mutant mice have been described previously [9 (link),10 (link),14 (link)], which were backcrossed with C57BL/6 mice for more than 10 generations. Mice were maintained on a standard rodent diet. Vps35 mutations were confirmed by genotyping using PCR and Western blot analysis. All experimental procedures were approved by the Animal Subjects Committee at the Georgia Regents University according to US National Institutes of Health guidelines.
Rabbit polyclonal anti-VPS35 antibody was generated using the antigen of GST-VPS35D1 fusion protein as described previously [9 (link),10 (link),14 (link)]. Rabbit polyclonal antibodies, including anti- ß-galactosidase (Cappel), anti-VPS26 (Abcam),anti-calretinin (Swant), and anti-melanopsin (Advanced Targeting Systems) antibodies, were purchased. Mouse monoclonal antibodies, including anti-neuronal class III ß-Tubulin (Tuj1,Convance), anti-neurofilament (DSHB), anti-calbindin D28k (Swant), anti-glial fibrillary acidic protein (GFAP, Chemicon), anti-rhodopsin (Abcam) and anti-myelin basic protein (MBP, Chemicon) antibodies, were also purchased. In addition, the chicken polyclonal anti- ß-galactosidase antibody (Abcam) and goat polyclonal anti-IBA1 antibody (Abcam) were used. Secondary antibodies were purchased from Jackson Immuno Research Laboratories, Inc. Other chemicals and reagents used in this study were of analytical grade.

Cells were washed twice with ice-cold PBS and then lysed in protein lysate buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1% [wt/vol] NP-40) and 1:7 protease inhibitor (Complete tablets; Roche) on ice for 30 min before storage at −70°C. After thawing, lysates were centrifuged at 16,100 × g for 5 min and the pellet was discarded. A bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce) was used to calculate protein concentrations according to the manufacturer's protocol, and equal amounts of protein were loaded for Western blotting, which was carried out as described previously (24 (link)). The primary antibodies used were rabbit anti-VPS52 (catalog no. ARP57644_P050; Aviva System Biology), mouse anti-actin (catalog no. 3700S; Cell Signaling), and anti-VPS35 (catalog no. Ab57632; Abcam), anti-VPS26 (catalog no. Ab23892; Abcam). The secondary antibodies were DyLight 680 and 800 (Cell Signaling).