Functional Characterization of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) N- and C-Terminal Domains during Xenopus laevis Development
Figure 2
Confirmation of full-length, N-terminal, or C-terminal TIMP-1 constructs overexpressed in X. laevis embryos. 4 ng of mRNA coding for full-length (T1FL), N-terminal (T1N), or C-terminal (T1C) TIMP-1 constructs was microinjected into X. laevis embryos at the 1-cell stage. (a) Overexpression of TIMP-1 mRNA as shown by RT-PCR analysis. RNA was isolated from stage 15 embryos. Control (uninjected) embryos expressed TIMP-1 at very low levels. Primers specific to each construct were used to confirm mRNA levels of T1FL (678 bp), T1N (453 bp), and T1C (252 bp). RT-PCR of EF1α was used as a loading control. (b) Increased levels of TIMP-1 constructs as shown by Western blot analysis. All constructs are HA tagged. Protein was isolated from stage 30 embryos. Anti-HA antibodies were used to confirm expression of each construct at the protein level (T1FL = 26 KDa, T1N = 18 KDa, and T1C = 12 KDa). No HA was detected in control uninjected embryos. β-actin was used as protein loading control.
