Kinase experiment:

To determine the time course of transglutamination, thymosin β4 (120 μM) is incubated with Dansylcadaverine (5 mM) in 70 μL buffer consisting of 10 mM Tris-HCl, pH 7.4, 15 mM CaCl2, 3 mM DTT. The reaction is started by addition of 0.1 U transglutaminase. Immediately after addition of the enzyme (t=0) and at indicated times,10 μL are taken from the mixture, diluted in 490 μL 0.1% TFA to stop the reaction and analyzed by HPLC[1].

Cell experiment:

MCF7 cells (2.4×104) are seeded into 35 mm plates. After 24 h incubation, CuO NPs are added with an increasing concentration in the presence or absence of 3-Methyladenine (3-MA) for different time periods . The cells are then incubated with 50 mM Dansylcadaverine (MDC) at 37℃ for 15 min and washed with 1×PBS three times with 5 min interval. Finally, the cells are observed under a fluorescence microscope[2].

Dansylcadaverine is a fluorescent probe.

Detection of Dansylcadaverine fluorescence after SDS-PAGE shows that the amount of actin-thymosin β4 crosslink is very low in the presence of Dansylcadaverine and transglutaminase, while there are large amounts of Dansylcadaverine-labeled thymosin β4 and actin. The amount of cross-link further decreases in the presence of Dansylcadaverine competing for Lys residues of either G-actin or thymosin β4 (100 μM). In the absence of thymosin β4, Dansylcadaverine-labeled actin polymerized to F-actin as revealed by fluorescence detection after native PAGE[1].

[1]. Huff T, et al. Thymosin beta(4) serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescentdansylcadaverine does not abolish interaction with G-actin. FEBS Lett. 1999 Dec 24;464(1-2):14-20. [2]. Laha D, et al. Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7. Biochim Biophys Acta. 2014 Jan;1840(1):1-9.