2OH-BNPP1

Kinase experiment:

In vitro kinase assays are performed using 200 ng of protein (BUB1-WT, or a kinase inactive mutant BUB1-KD), and 2 µg of substrate (TGFBRI-WT, TGFBRI-KD, SMAD3 or H2A) in 1X kinase buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2, 1% (v/v) Glycerol, 0.1% (v/v) Triton X 100, DTT, PMSF, Na3VO4 (1 mM each), 2 mM NaF and β-glycerol phosphate) in 20 µL volume containing 10 µCi 32p-ATP and 300 µM cold ATP. Reactions are run at 30ºC for 0.5-1 hour and quenched using Laemmli buffer and resolved using a 4-12% Bis-Tris gel. Quantitative autoradiography is performed using a Typhoon FLA 9000 scanner.

Animal experiment:

Cell line-derived xenografts are established by implanting 2.5×106 A549 cells subcutaneously into each flank of 4-6 week old male SCID mice. When tumors reach a volume between 40 to 60 mm3, mice are injected with a single intraperetoneal dose of SB431542 (10 mg/kg body weight), a dose previously reported to inhibit TGFβ signaling in mouse models, or 2OH-BNPP1 (50 mg/kg), or vehicle (DMSO). Tumors are excised 4 hours after treatment and fixed in formalin. Paraffin-embedded sections are stained using an antibody for phosphorylated SMAD2, and micrographs are taken with an Olympus microscope fitted with an Olympus DP-70 high resolution digital camera. The proportion of nuclei that stains positive for phosphorylated SMAD2 are counted in three random fields per tumor per treatment condition [vehicle (n=4), SB431542 (n=2), and 2OH-BNPP1 (n=5)]. A two -ided Student's t-test is performed to assess statistical significance. Slides are adjusted for brightness and contrast with Adobe Photoshop CS2, but the micrographs undergo no further manipulations.

References:

[1]. Nyati S, et al. The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling. Sci Signal. 2015 Jan 6;8(358):ra1.