Brefeldin a b7651

Nocodazole (M1404), PKH67 (green) (PKH67GL-1KT), PKH26 (red) (PKH26GL-1KT), brefeldin-A (B7651, used at 5 μg/ml), N-ethylmaleimide (E3876, used at 50 μM), LiCl2 (L-4408), PMA (used at 100 nM; P8139), and dynasore (D7693, used at 100 μM) were purchased from Sigma (St. Louis, MO, USA). TNF-α (210-TA) (used at 17 ng/ml), VEGF (used at 19 ng/ml; 293-VE), and neutralizing antibodies to VEGF (used at 1 μg/ml; MAB293) and VEGFR2 (used at 1 μg/ml; MAB3572) were purchased from R&D Systems (Minneapolis, MN, USA). Smac mimetic TL-32711 (A-1901) (used at 100 nM) was purchased from Active Biochem (Maplewood, NJ, USA). Cisplatin (NDC 0015-3220-22) was purchased from Bristol Laboratories (Princeton, NJ, USA). Antibodies to VEGF (for WB: Sc-7269), PKC-ζ (Sc-17781), K-Ras (Sc-30), and VEGFR2 (for WB: Sc-504) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to E-cadherin (3195), p70S6K (2708), phospho-S6 ribosomal protein (Ser-235/236) (2211), S6 ribosomal protein (2317), syntaxin-6 (2869), and cdc42 (2466) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to β-catenin (610153) was purchased from BD Transduction Laboratories (Lexington, KY, USA). BI-D1870 (used at 5 μM; S2843) was purchased from Selleckchem (Houston, TX, USA).

The RCC4 cell line was a gift from Denise Chan (University of California, San Francisco, San Francisco, CA) and was authenticated by DDC (DNA Diagnostics Center) Medical using the short tandem repeat method and tested to be mycoplasma-free in November 2015. 786-O cells were purchased from the Cell Culture Facility at Duke University (Durham, NC, USA). 786-O cell line has been authenticated by STR DNA profiling and tested to be mycoplasma-free by Duke Cell Culture Facility. All cells were cultured in DMEM with 4.5 g/L glucose and 4 mM Glutamine (11995-DMEM, ThermoFisher Scientific) and 10% heat-inactivated fetal bovine serum (Hyclone # SH30070.03HI) in a humidified incubator, at 37 °C with 5% CO₂. Tunicamycin (T7765) and brefeldin A(B7651) were purchased from Sigma. Erastin was synthesized at Small Molecule Synthesis Facility at Duke University.