TG activity in preadipocytes arises from FXIII-A. (A) In situ assessment of TG activity during differentiation of 3T3-L1 preadipocytes shows a significant increase upon induction of differentiation until day 2, which is followed by a gradual decrease as cells begin to accumulate lipids. TG activity was assessed by BPA incorporation assay (n = 3). Statistical analysis was done using analysis of variance *P < .05; ***P < .001. Error bars represent SEM. (B) Immunofluorescence tracking of TG activity using NC9, which incorporates irreversibly into the active TG enzyme. Immunofluorescence microscopy shows colocalization (merge, yellow) of NC9-dansyl (green) and FXIII-A (red), identifying FXIII-A as the active TG enzyme in preadipocytes. Nuclei are visualized with 4,6 diamidino-2-phenylindole (DAPI) (blue). Color correlation distribution, constructed using the Color Inspector 3D plug-in of Image J, shows the extent correlation of colocalization (n = 2). Scale bar represents 10 µm. (C) TG activity is located on the cell surface. Cell-surface biotinylated samples were affinity purified using Neutravidin beads and subsequently detected with dansyl antibody, which showed a major band >150 kDa and a weaker band between 75 and 50 kDa (n = 3). (D) Immunoprecipitation of NC9-labeled material with anti-dansyl antibody and detection with anti-mouse FXIII-A antibody positively identified the active TG enzyme to be FXIII-A (running >150 kDa) (n = 5). (E) WB detection of FXIII-A in 3T3-L1 preadipocytes and MEFs before (D0) and after induction of differentiation (DM) (day 1) using anti-mouse FXIII-A antibody. HMW FXIII-A was observed at day 1 of differentiation (n = 3). (F) WB detection of nonactivated human FXIII (hFXIII), activated human FXIII (Act.hFXIII), mouse platelet-rich plasma (mPRP), and 3T3-L1 cell extract using anti-human FXIII-A antibody (A-4). WB shows human and mouse FXIII-A at different molecular weights, with mouse FXIII-A being smaller (n = 3). (G) WB detection of nonactivated human FXIII (hFXIII), activated human FXIII (Act.hFXIII), and mPRP using anti-human FXIII-A antibody (ab97636). WB shows detection of 2 FXIII-A bands in mPRP, one at 75 kDa and a smaller band between 50 and 75 kDa (n = 3). (H) A smaller FXIII-A band is active as a TG enzyme. Mouse platelet lysate (mPlts) and mouse plasma (mPlasma) were activated with thrombin and Ca²⁺ for 1 hour at 37°C and further incubated with NC9. Dansyl incorporation into the active enzyme was visualized by WB detection of dansyl. Dansyl was found to be integrated into a band between 50 and 75 kDa, which represents the smaller form of FXIII-A (n = 3).