Figure 5
TG2 levels and location during early adipogenesis and in WAT. (a) Western blot analysis of total cell lysate from Tgm2+/+ MEFs during adipocyte differentiation. TG2 levels remain constant with no major fluctuations during differentiation. Actin used as loading control. (b) Transamidase activity in Tgm2−/− and Tgm2+/+ MEFs during adipogenesis was assessed in situ using 5-(biotinamido) pentylamine as an activity probe. Graph displayed is biotin detection in cells and the activity is normalized to TG-activity on day 0 of Tgm2+/+ MEFs (set for 100%). Results are mean values±SEM (n=3). (c) Western blot analysis of cell surface biotinylated protein extract for TG2 protein levels in Tgm2+/+ MEFs during adipocyte differentiation. TG2 levels on cell surface increase with initiation of differentiation (day 1). (d) Immunofluorescence staining of TG2 (green) during early differentiation of Tgm2+/+ MEFs. Nuclei were stained with DAPI (blue). Cells not treated with Triton-X100 show the extracellular distribution of TG2; Triton X-100 permeabilized cells show intracellular distribution; Scale bar equals 100 μm. (e) Immunofluorescence staining of whole-mount mouse white adipose tissue (WAT) showing distribution of TG2 (red) and lipids (Bodipy 493/503, green) in the tissue; TG2 is mainly extracellular; Scale bar equals 50 μm
