Mouse anti ve cadherin sc 9989

After the cells were incubated with BSA or TAGE for 48 h, immunofluorescence staining was performed as previously described^{45 (link)}. Cells grown on a Nunc Lab-Tek II chamber slide (154534PK; Thermo Fisher Scientific) were removed from the medium and fixed in 95% ethanol for 1 h at room temperature. Cells were rinsed with PBS (−) and incubated with 0.5% Triton X-100 in PBS (−) for 5 min at room temperature. The cells were then blocked for 5 min using Blocking One Histo (06349-64; Nacalai Tesque Inc.) before being incubated with mouse anti-VE-cadherin (sc-9989 at 1:2000; Santa Cruz Biotechnology, Inc.) or rabbit anti-ZO-1 (GTX108627 at 1:1000; GeneTex) antibody in Can Get Signal Immunostain Immunoreaction Enhancer Solution B (NKB-601; Toyobo) for 1 h. Subsequently, the cells were rinsed three times with PBS-T and incubated with Cy5 conjugated goat anti-mouse IgG (AP200S at 1:1000; Chemicon International) or Cy3 conjugated goat anti-rabbit IgG (AP132C at 1:2000; Chemicon International) in Can Get Signal Immunostain Immunoreaction Enhancer Solution B for 1 h. After rinsing three times with PBS-T, the coverslips were mounted on glass slides using a ProLong Glass Antifade Mountant with NucBlue (P36983; Thermo Fisher Scientific), and confocal microscopy was performed using a scanning confocal microscope system (LSM 800; Carl Zeiss).

For immunofluorescence and Western blotting, the following primary antibodies were used: rabbit anti-p62 [P0067] (1:2,000 for Western blot; 1:100 for immunofluorescence), mouse anti-β-actin [A1978] (1:10,000), rabbit anti-LC3B [L7543] (1:1,000), rabbit anti-CCM2 [HPA020273] (1:1,000), and rabbit anti-AMBRA1 [PRS4555] (1:1,000) from Sigma-Aldrich; rabbit anti-GAPDH [#2118] (1:5,000), rabbit anti-mTOR [#2983] (1:1,000), rabbit anti-phospho-mTOR (Ser 2448) [#5536] (1:1,000), rabbit anti-p70 S6 Kinase [#9202] (1:1,000), rabbit anti-phospho-p70 S6 Kinase (Ser 371) [#9208] (1:1,000), rabbit anti-4E-BP1 [#9644] (1:1,000), rabbit anti-phospho-4E-BP1 (Thr 37/46) [#2855] (1:1,000), rabbit anti-ULK1 [#8054] (1:1,000), and rabbit anti-phospho-ULK1 (Ser 757) [#6888] (1:500) from Cell Signaling; rabbit anti-phospho-AMBRA1 (Ser 52) [#ABC80] (1:1,000) from Millipore; rabbit anti-alpha-SMA [NB 600-531] (1:1,000) from NovusBio; mouse anti-N-cadherin [33–3900] (1:500) from Invitrogen; goat anti-CD31/Pecam-1 [sc-1506] (1:1,000), mouse anti-LAMIN A/C [sc-7292] (1:1,000), and mouse anti-VE-cadherin [sc-9989] (1:500) from Santa Cruz Biotechnology; and rabbit anti-KRIT1 (1:500) from S.F. Retta.