Fig. 1
Identification of wild-type TFF2 and fusion TFF2-CTP-Flag protein delivered by Ad-Tff2 and Ad-Tff2-CTP-Flag in the blood. a TFF2 level assayed by ELISA in the blood from naive wild-type and CD2-Tff2 transgenic mice and mice at time point 6 months after induction of tumorigenesis with AOM/DSS treatment. Dunn’s multiple comparisons test after one-way ANOVA test, ns, non-significant, *p < 0.05, ****p < 0.001. b Amino acid sequence of fusion protein TFF2-CTP-Flag: TFF2 amino acid sequence—bold regular, CTP—regular, S—underlined, original cysteine substituted for serine, additional amino acids residues -bold italic shadow, Flag-underlined regular, Stop—stop codon. c Schematic presentation of fusion construct Tff2-2CTP-3Flag. d Map of GV314 vector with inserted TFF2 gene. e–j Time course of TFF2 and TFF2-CTP-Flag level after single administration of adenoviruses Ad-Tff2 and Tff2-CTP-Flag. Tff2-null mice were injected with 5 × 108 pfu of Ad-Tff2 or Ad-Tff2-CTP-Flag and sacrificed at indicated times post infection. Time-course of Tff2 mRNA (e) and Tff2-CTP-Flag mRNA (h) expression in the liver. Total mRNA was isolated from the liver and then Tff2 mRNA was detected by qPCR, fold of change normalized on housekeeper Hprt mRNA. For each time point 3 mice were used, graphed as mean ± SD. f, i Kinetic of TFF2 protein and TFF2-CTP-Flag fusion protein in the blood assessed by ELISA and expressed as mean ± SD at each time point, three animals per group. g, j western blot for TFF2 and TFF2-CTP-Flag in mouse serum. Five microliter of mouse serum were loaded in each lane and western blot was developed with antibody produced against C-end of TFF2 molecule, arrows indicate the position of TFF2 and TFF2-CTP-Flag with calculated sizes 12 and 25–26 kDa accordingly. k Western blot of blood sample (5 µl) from Tff2-null mouse taken on day 7 after Ad-Tff2-CTP-Flag administration. Western blot was developed with anti-Flag antibody; arrows indicate the positions of TFF2-CTP-Flag fusion under reduced and non-reduced conditions
