Ab97545

Antibodies used in this study are as follows: SNX1 [clone 51; 611482; BD Bioscience; immunofluorescence (IF) 1:200], GLUT1 (ab40084; Abcam; IF 1:200), Golgin97 (clone CDF4; A-21270; Thermo Fisher Scientific; IF 1:400), VPS26 (ab23892; Abcam; IF 1:200), VPS35 (ab10099; Abcam; IF 1:200), VPS35 [ab97545; Abcam; IF 1:200), VPS35 [ab157220; Abcam; western blotting (WB) 1:1000], VPS29 (ab98929; Abcam; WB 1:200), FAM21 (gift from Daniel D. Billadeau, Mayo Clinic, Rochester, MN; IF 1:400), EEA1 (N-19; sc-6415; Santa Cruz Biotechnology; IF 1:200), TGN46 (AHP500G; Bio-Rad Laboratories; IF 1:200), anti-Myc (gift from Harry Mellor, The University of Bristol, UK; IF 1:500), LAMP1 (DSHB Hybridoma Product; H4A3; deposited to the DSHB by August, J.T./Hildreth, J.E.K.; IF 1:500), LAMP1 (ab24170; Abcam; IF 1:200), mouse EEA1 (610457; BD Bioscience; IF 1:200), CI-MPR (ab124767; Abcam; WB 1:1000, IF 1:200), Itgβ1 (TS2/16; IF 1:200), SNX6 (Clone D-5; 365965; Santa Cruz Biotechnology; WB 1:500), PMP70 (PA1-650; Invitrogen; IF 1:200), WASH1 (gift from Daniel D. Billadeau; IF 1:400), C16orf62 (PA5-28553; Pierce; IF 1:200), sortilin (ab16640; Abcam; WB 1:1000), cathepsin D (21327-1-AP, Proteintech; WB 1:1000), SNX5 (ab180520; Abcam; WB 1:500) and β-actin (A1978; Sigma-Aldrich; WB 1:2000).

Proteins from total cell lysates (10 to 20 μg) were separated by SDS-PAGE, transferred onto PVDF membranes (Hybond 0.2 μm; Amersham GE Life Sciences), and probed using the indicated antibodies. The secondary HRP-conjugated antibody was visualized by chemiluminescence (SeraCare developer solution). For anti-tubulin probing, membranes were first treated with stripping buffer (Thermo Scientific), washed, and reprobed. Polyclonal rabbit antibodies against VPS35 (ab97545; abcam), VPS29 (ab98929; abcam), rabbit monoclonal antibody against VPS26 (ab98929; abcam) or mouse monoclonal antibody against β-tubulin (catalog no. T8328; Sigma) were used according to the manufacturer’s instructions. Quantification of immunoblots was performed using ImageJ.