Cathepsin d

Tissues were lysed in 1 mL ice-cold PRO-PREP buffer (iNtRON, Seongnam, Korea). Protein concentrations in supernatants were evaluated with a BCA protein assay kit (Thermo Scientific, South Logan, UT, USA). Protein (30 μg/lane) was electrophoresed on 10–15% SDS gel, and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat dry milk for 2 h at RT and then incubated with rabbit primary antibodies against β-actin (1:1000, Santa Cruz Biotechnology), LC3-II, and p62 (1:1000 and 1:1000, respectively, Sigma-Aldrich, St. Louis, MO, USA), ATP6E, cathepsin D, and caspase-3 (1:1000, 1:1000 and 1:500, respectively, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. Membranes were incubated with HRP-conjugated anti-rabbit IgG secondary antibodies (1:1000, Abfrontier Co., Ltd., Seoul, Korea) for 2 h at RT. The protein bands were visualized using a chemiluminescence detection kit (Thermo Scientific, South Logan, UT, USA). The same membranes were subsequently used for β-actin immune detection, and equal protein loading was ensured. The optical density for quantification was obtained using Gel-Pro Analyzer version 3.1 (Media Cybernetics, Silver Spring, MD, USA).

Antibodies included synaptotagmin-1 (1:1000; #14558S Cell Signaling Technology, Danvers, MA), Cathepsin-D (1:1000, #69854, Cell Signaling Technology), Actin (1:1000; #4967, Cell Signaling Technology) and GFAP (1:5000; #ab190288, Abcam, Waltham, MA).

HUVECs and HAECs were purchased from Lonza (Walkersville, MD, USA) and cultured with an endothelial cell growth medium-2 (EGM-2) bullet kit (Lonza) without addition of vascular endothelial growth factor^{21 (link)}. HUVECs were cultured in cell culture dishes precoated with 0.2% gelatin (Sigma-Aldrich) and used at passages 4 to 6. Antibodies against phospho-Beclin-1(Ser93), Beclin-1, p62, LC3B, Cathepsin D, STX17, cleaved caspase-8, and cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against VAMP8, SNAP29, and YKT6 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The STX7 antibody was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA). Antibodies against α-tubulin and β-actin were purchased from Sigma-Aldrich. Bafilomycin A1 was purchased from Sigma-Aldrich. Z-IETD-FMK were purchased from abcam (Cambridge, MA, USA).

Western blotting analysis was performed as previously described [73 (link)]. Sources of antibodies are: BAX, BNIP3, Mcl-1 and acid ceramidase; Bcl-xL, Bcl-2, Beclin-1 and Cytochrome C: BD Biosciences (San Diego, CA); BAK, BOK, BIM, BAD, PUMA, xIAP and Cathepsin D: Cell Signaling Tech (Danvers, MA); Cathepsin B: Abcam (Cambridge, MA); β-actin: Sigma-Aldrich (St Luis, MO).