Sab2702192

The following antibodies were used: β-ACTIN mouse monoclonal (Sigma-Aldrich, A5316; 1:8000 WB), LAMP-1 rabbit polyclonal (Abcam, ab24170, 1:500 IF), LAMP-1 mouse monoclonal (DSHB, H4A3; 1:500 WB), and MYC mouse monoclonal (Sigma-Aldrich, SAB2702192; 1:2000 WB; 1:200 IF).

Cell samples from each well plate were independently homogenized and solubilized in 100 µl of Tris-Glycine SDS sample buffer (Novex Life Technologies) supplemented with complete protease inhibitor cocktail (Roche). Extracted proteins were subjected to SDS-PAGE (Mini-Protean-III, Bio-Rad) using 15% polyacrylamide gels, blotted onto PVDF membranes (Hybond-P, GE Healthcare) and probed with mouse monoclonal anti-FLAG M2 (F3165, Sigma–Aldrich, 1:15000) or anti-Myc-tag (SAB2702192, Sigma–Aldrich, 1:1000) antibodies, followed by incubation with anti-mouse IgG peroxidase antibody (A9917, Sigma–Aldrich, 1:33000). Immunological signals were detected by Super-Signal West Pico PLUS chemiluminescent substrate (Thermo Scientific). Protein extracts from nontransfected cells were used as negative controls. The SeeBlue Plus2 prestained protein standard (Invitrogen) was included in each membrane. Equivalence of protein loading was confirmed by Amido Black 10B staining of blots after immunodetection. Western blot films (Amersham Hyperfilm ECL, GE Healthcare) were scanned with the GS-800 Calibrated Densitometer and analyzed with the image analysis program Quantity One (Bio-Rad). Densitometric analysis was performed as described in the previous Methods subsection.

Hela cells transiently transfected with Myc-tagged SORD were cultured in glass-bottomed dishes. Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized, and blocked in PBS containing 0.1% TritonX-100 (Beyotime Biotechnology, China) and 5% bovine serum albumin (Sigma, USA) for 60 min. Cells were then incubated with anti-Myc (1:1000, SAB2702192, Sigma, USA) antibody at 4 °C overnight, followed by secondary anti-mouse IgG Alexa Fluor 488 antibodies (1:1000, Life Technologies, USA). Cell nuclei were then stained with 40, 6-diamidino-2-phenylindole (DAPI; 1:5000, Life Technologies, USA). Fluorescence images were captured by Olympus FV3000 confocal system (Olympus Corporation, Japan).