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. Author manuscript; available in PMC: 2008 Oct 28.
Published in final edited form as: FASEB J. 2008 May 23;22(8):3010–3023. doi: 10.1096/fj.07-100966

Fig. 4.

Fig. 4

Ang1-256 Monomers are Expressed in Heart. (A) Recombinant truncated ang1-256 has a modified influenza hemagluttinin secretion signal (HS) and FLAG (F) tag fused to ang1 at amino acid 256. Ang1-256 has 6 amino acids of the coiled coil (C), linker (L) and fibrinogen-like domains of ang1 followed by C-terminal V5 and 6X polyhistidine (H) tags. (B–C) Ang1-256/pcDNA (ang1-256) or pcDNA control (Con) were retroorbitally injected into C57 mice (studies done in triplicate). (B) LV ang1-256 mRNA was detected at all timepoints. cDNA synthesis reactions were conducted ± reverse transcriptase (RT) with ang1-256/pcDNA plasmid (positive control). (C) In mouse heart, ang1-256 protein was detected at all timepoints using fluorescent immunohistochemistry/confocal imaging. Ang1-256 was labeled using FLAG antibody linked to AlexaFluor647 and nuclei labeled (DAPI). Negative controls included substituting rabbit IgG for primary antibody and injecting mice with pcDNA vector (control) (1 d). (D) HEK293 cells were transfected with ang1-256/pcDNA. Ang1-256 proteins were purified by anti-FLAG M1 affinity column chromatography (Sigma), dialyzed, and concentrated. Western blots probed with V5 antibody show ang1-256 monomer protein at the predicted MW (34 kDa) in nonreduced (NR) and reduced (R) conditions.