The distribution of TG2 in nuclear fractions of maturing NB4 cells. (A) Localization of TG2 by confocal microscopy. NB4 promyelocytes were cultured in the presence of 1 μM ATRA for 6 days. (i-iii) Cells were fixed, permeabilized, and labeled for TG2 with monoclonal antibodies followed by Alexa Fluor 633–tagged GAMIG (i, blue), and were also stained with PI (ii, red). Panel iii is the overlay image. Note that TG2 is present in the nucleus. In the control, unspecific labeling by Alexa Fluor 633-GAMIG was checked by omitting anti-TG2 antibody (iv). Panel v shows the nucleus of the same control cell, and panel vi, the overlay image of panels iv and i. The thickness of the optical sections shown is 800 nm. (B-D) Extraction of TG2 from nuclei of NB4 cells treated with ATRA. Nuclear samples were fractionated into (B) Triton X-100, (C) NP40-soluble, and (D) nuclear matrix compartments as described in “Materials and methods.” Briefly, the Triton X-100–soluble lipids and proteins were extracted, and then NP40-soluble fractions were obtained. The remaining insoluble protein remnant was suspended in nuclear buffer and sonicated for a short time to get the soluble DNA and protein fraction. Last, with the help of 5 M NaCl, the high salt–soluble protein was extracted from this suspension. The last 2 fractions contained mostly the nuclear matrix, chromatin, and associated proteins.32 From each fraction, 25 μg protein was analyzed by Western blotting using monoclonal antibody to detect TG2. To confirm that the fractions were free of cytoplasmic contamination and that they contained a different protein pattern, the blotted membranes were stained by Coomassie blue and immunoblotted for β-tubulin (not shown).
