Video-Rate Bioluminescence Imaging of MMP-2 Figure 4. Characterization of MMP2-GLase expressed in HeLa cells with immunochemical analyses. MMP2-GLase expressed in HeLa cells was identified by Western blot (A and B) and immunofluorescence (C) analyses. (A) Western blot analysis of MMP2-GLase in cell lysate using anti- MMP-2 and anti-GLase antibodies (MMP-2 and GLase for lane 1 and lane 2, respectively). HeLa cells were transfected with pcDNA3-hMMP2-GLuc for the expression of MMP2-GLase (MMP2-GLase) and cultured for 24 h, and proteins in the cell lysate were separated in a 5–20% gel. The numbers on the left margin represent the molecular weight of size maker proteins (XL ladder, Promega). The molecular sizes of two bands by arrow 1 and 2 correspond to the pro-form (89.4 kDa) and processed form (80.3 kDa) of MMP2-GLase, respectively. (B) Western blot analyses of the cell lysate and the conditioned medium expressing MMP2-GLase, MMP2-FLAG, and wild type MMP-2 using anti-MMP-2 antibody (MMP-2). HeLa cells transfected with pcDNA3 (as a control vector: lane 1 and 2), pcDNA3-hMMP2 (lane 3 and 4), pcDNA3-hMMP2-Flag (lane 5 and 6), or pcDNA3-hMMP2-GLuc (lane 7 and 8) (Cont, MMP-2, MMP2-FLAG, and MMP2-GLase, respectively) were cultured for 24 h, and further cultured for 24 h in HBSS. Proteins in the cell lysate (lane 1, 3, 5, 7) and the concentrated proteins from the conditioned medium of HBSS (lane 2, 4, 6, 8) were separated in a 10% gel. The numbers on the left margin represent the molecular weight of size maker proteins (Precision Plus Protein All Blue standards, Bio-Rad). Two bands indicated by arrow 1 and 2 at the right margin correspond to pro- and processed forms of MMP2-GLase, respectively. Two bands indicated by arrow 3 and 4 at the right margin correspond to pro- and processed forms of MMP-2, respectively. Endogenous pro- and processed MMP-2 (71.0 kDa and 62.0 kDa) slightly detected in lane 1 and 2, exogenous pro- and processed MMP-2 in lane 3 and 4, and pro- and processed MMP2-FLAG (72.2 kDa and 63.2 kDa) in lane 5 and 6. (C) Immunofluorescence images of MMP2-FLAG (Green) and MMP2-GLase (Magenta) co-expressed in HeLa cells. Overlay; Merged image of the both fluorescence images. IB, immunoblot; TF, transfection; DF, dye front. doi:10.1371/journal.pone.0025243.g004 S4). These results suggested that inactive pro-MMP-2 secreted from the under membrane at the leading edge and then bound to specific sites on the bottom side of migrating cells (Figure 8). The specific luminescence spots of MMP-2 on the cell surface might indicate the micro domains for activation of pro-MMP-2 or for binding of active MMP-2. Our video-rate imaging method using the fusion protein of MMP2 with GLase can be applied to investigate molecular mechanism for exocytotic secretion of PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25243