Halt protease and phosphatase inhibitor single use cocktail

For the validation of protein targets, two sets of 48 tiling probes were used to affinity purify mature SAMMSON and HPRT transcripts (Biosearch Technologies). Briefly, 100 µL of Streptavidin Sepharose High Performance beads (GE Healthcare) were coupled to 400 pmol of biotinylated probes against SAMMSON overnight at 4 °C. Cells (6 × 10⁷ cells per sample) were lysed in 2 mL of pull-out buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 2.5 mM MgCl₂, 0.05% Igepal, 60 U mL^-1 Superase-In (Ambion), 1 mM dithiothreitol (DTT) and 1× Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies)) and incubated for 3 h with the beads at 4 °C on a rotating wheel. As a negative control, an additional sample was digested with 10 µg mL^-1 RNase A for 10 min at room temperature before incubation with SAMMSON probes.
For the crosslinking experiments, cells were washed once in PBS, crosslinked dry at 400 mJ cm^-1 and lysed. Washes were performed in RNase free water.

Cells were treated for 24 h with DMSO (as vehicle), TF1, TF2a, TF2b or TF3 (20 μM) in 60 mm dishes, and then lysed in mammalian protein extraction reagent supplemented with Halt^™ Protease and Phosphatase Inhibitor Single-Use Cocktail (LifeTechnologies, Grand Island, NY, USA). The concentration of protein was measured using a BCA Protein Assay Kit (Thermo, Waltham, MA, USA). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membrane was blocked with 5% non-fat milk in Tris-buffer saline containing 0.1% Tween-20 for 1 h at room temperature and incubated with the indicated primary antibodies overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibody for 2 h at 37°C. Detection was performed by SuperSignal West Dura Extended Duration Substrate (Life technologies) and ChemiDoc^™ MP System (Bio-Rad, Hercules, CA, USA). Protein bands were quantified with NIH ImageJ software (NIH), normalized by corresponding GAPDH for analysis.

Cells were seeded in 60-mm dishes at 1×10⁶ cells/dish, incubated overnight, and treated with TF3, CDDP or the combination for 24 h. Then cells were harvested with M-PER Mammalian Protein Extraction Reagent (Pierce, St Louis, MO, USA) supplemented with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies, Grand Island, NY, USA). The total protein levels were detected with BCA Protein assay kit (Pierce, St Louis, MO, USA). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membrane was blocked with 5% skim milk in Tris-buffer saline containing 0.1% Tween-20 for 1 h at room temperature, and then incubated with specific primary antibodies and appropriate secondary antibodies conjugated with horseradish peroxidase. The antigen-antibody complex was visualized with Super Signal West Dura Extended Duration Substrate (Life technologies, Grand Island, NY, USA) and ChemiDoc™ MP System (Bio-Rad, Hercules, CA, USA). Protein bands were quantitated with NIH Image J software and normalized by GAPDH bands for analysis.

After treatments, cells were lysed using M-PER Mammalian Protein Extraction Reagent (Pierce) supplemented with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies). The protein levels were determined using BCA Protein assay kit (Pierce). After boiling for 6 min in loading buffer (Bio-Rad), equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred onto a nitrocellulose blotting membrane. The membrane was blocked with 5% nonfat dried milk in Tris Buffered Saline (Bio-Rad) containing 0.1% Tween-20 (TBST) at room temperature for 1 h and subsequently incubated with specific primary antibodies overnight at 4 °C. The membrane was washed three times (10 min each) with TBST and then incubated with an appropriate secondary antibody conjugated with horseradish peroxide for 1 h at room temperature. After three 10 min washes with TBST, antigen-antibody complex in each blot was visualized with Super Signal West Dura Extended Duration Substrate (Life technologies) and ChemiDoc™ MP System (Bio-Rad). Protein bands were quantitated with NIH Image J software and normalized by GAPDH bands for analysis.