Irdye infrared secondary antibody

Proteins were extracted, sonicated, centrifuged at 13,000 rpm for 45 min at 4 ^oC, and supernatants used for immunoblot analysis, as previously described17 (link)18 . Actin was always used as an internal loading control. Primary antibodies used were as follows: anti GSAP full length (1:200) and GSAP-16 kDa (1:150) (Thermo Scientific); anti-5LO (1:500) (BD Bioscience); anti-12-15LO (1:200) (Santa Cruz Biotech., Dallas, TX); anti-TMP21 (1:200), anti-CD147(1:200), anti-caspase-3 (1:200) (Santa Cruz Biotech., Dallas, TX); anti-β-actin (1:200) (Santa Cruz Biotech., Dallas, TX). IRDye infrared secondary antibodies were from LI-COR Bioscience (Lincoln, NE).

Adherent and detached cells were harvested by scraping, washed in PBS and lysed in RIPA buffer containing protease inhibitors (Thermo Scientific). For studies of tubulin, cells were harvested in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaC1, 1mM EDTA, 1% Triton X-100 with protease and phosphatase inhibitors), frozen and sonicated to clarify. Protein concentrations were determined by Bradford assay using Coomassie Protein Assay Reagent (Thermo Scientific). Samples were separated by SDS-PAGE and transferred to nitrocellulose or PVDF. The following antibodies were used: mouse anti-Mcl-1, rabbit anti-Bak, and rabbit anti-Bax (Abcam); mouse anti-β-actin (Thermo Scientific, BA3R); rabbit anti-cleaved PARP Asp214, rabbit anti-XIAP and rabbit anti-Bcl-XL (Cell Signaling Technologies), mouse anti-human CD71 (transferrin receptor) PE conjugate (Biolegend), mouse anti-acetylated tubulin (Sigma, T7451), mouse anti-a-tubulin (Sigma, T9026), and GAPDH (Millipore, 6C5). HRP-conjugated mouse and rabbit secondary antibodies (Santa Cruz Biotechnologies or Cell Signaling Technologies) were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Alternatively, IRDye infrared secondary antibodies (LiCor) were utilized and blots scanned on the Odyssey (LiCor).

HEK293 cells were lysed on ice for 60 min in lysis buffer containing 10 mM Tris (pH 7.4), 1% Triton X-100, 150 mM NaCl, 10% glycerol, and freshly added protease inhibitors (Roche Complete Protease Mini, Cat # 4693159001) and phosphatase inhibitors (PhosStop pellets, Sigma Aldrich, Cat # 4906845001). Cell lysates were centrifuged at 15,000 × g for 20 min at 4°C. Supernatant was collected as the soluble fraction. The pellet was washed 3 times with lysis buffer and centrifuged at 15,000 × g for 5 min each at 4°C. The pellet was resuspended in lysis buffer supplemented with 4% SDS, sonicated 3 times, boiled for 30 min, and collected as the insoluble fraction. Protein concentration was determined using Lowry Protein Assay (Bio-Rad). For Western blot analysis, proteins were subjected to 4%–12% bis-tris midi gels (Bio-Rad) and transferred to nitrocellulose membrane. Membrane was blocked with Odyssey Blocking Buffer (Fisher Scientific). The following primary antibodies were used: mouse anti-Myc (Cell Signaling, Cat # 2276, 1:1000 dilution) and mouse anti-beta tubulin (Sigma-Aldrich, Cat # T8328, 1:8000 dilution). Appropriate IRDye infrared secondary antibodies were purchased from LI-COR Biosciences and used at a dilution of 1:10000. Odyssey Infrared Imaging System (LI-COR Biosciences) was used to detect the signal of target proteins.