Volume 114, Issue 4, Pages 706-713 (April 1998) Induction of mitogen-activated protein kinase signal transduction pathway during gastric ulcer healing in rats  Rama Pai, Masayuki Ohta, Rabiha M. Itani, I.James Sarfeh, Andrzej S. Tarnawski  Gastroenterology  Volume 114, Issue 4, Pages 706-713 (April 1998) DOI: 10.1016/S0016-5085(98)70584-0 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Total tyrosine kinase activity in the membranes of gastric specimens from sham-operated rats and in membranes from 1-, 3-, and 7-day ulcers. ct, controls. Membrane PTK activity was measured by phosphorylation of peptide substrate (RR-SRC) specific for tyrosine kinase29 in the presence of [γ-32P]ATP. Proteins were removed by acid precipitation, phosphorylated peptide was bound to a phosphocellulose disc, and radioactivity was measured by scintillation counting. Values are expressed as pmol/mg protein and represent mean ± SEM of six experiments performed in triplicate. *P < 0.001 vs. controls (sham-operated rats). Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 EGF-R levels in gastric specimens from sham-operated rats (□) and 1-, 3-, and 7-day ulcers (■). The detergent solubilized samples of gastric specimens containing 150-μg proteins were subjected to Western blot analysis using specific polyclonal antibody against EGF-R. Density of protein bands was analyzed using a laser densitometer. Values represent relative densities of EGF-R band (mean ± SEM; n = 6). ct, controls. *P < 0.05 vs. controls (sham-operated rats). Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Time-course changes in the extent of tyrosine phosphorylation of EGF-R (170-kilodalton band) and other mucosal proteins in gastric specimens from controls (sham-operated rats) and 1-, 3-, and 7-day ulcers. Tissue lysates containing 150-μg proteins were subjected to Western immunoblotting with monoclonal antiphosphotyrosine antibody. (A) Lanes represent tyrosine phosphorylated proteins from controls (lane 1), 1-day ulcer (lane 2), 3-day ulcer (lane 3), and 7-day ulcer (lane 4) specimens. (B) EGF-R was immunoprecipitated with polyclonal anti–EGF-R antibody, and the immunoprecipitate was subjected to immunoblotting with antiphosphotyrosine antibody. Lanes represent immunoprecipitates from controls (lane 1), 3-day ulcer (lane 2), and 7-day ulcer (lane 3) specimens. The Western blot is a representative of six separate experiments. This data clearly showed that phosphorylated 170-kilodalton band is indeed EGF-R. Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 ERK1 activity in gastric specimens from control (ct) (sham-operated) rats (□) and in 1-, 3-, and 7-day ulcers (■). The activity in tissue lysates was determined by measuring the levels of radiolabeled [γ-32P]ATP incorporated into MBP (pmol/mg protein) by ERK1 immunoprecipitated from cytosolic lysates. Values represent mean ± SEM of six experiments performed in triplicate. *P < 0.007 vs. controls (sham-operated rats). Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 ERK2 activity in gastric specimens from control (ct) (sham-operated) rats (□) and in 1-, 3-, and 7-day ulcers (■). The activity in tissue lysates was determined by measuring the levels of radiolabeled [γ-32P]ATP incorporated into MBP (pmol/mg protein) by ERK2 immunoprecipitated from cytosolic lysates. Values represent mean ± SEM of six experiments performed in triplicate. *P < 0.01 vs. control (sham-operated rats). Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Photographs showing gross appearance of 7-day ulcers after vehicle and Tyrphostin treatment. The stomach was opened along the greater curvature. Compared with vehicle-treated rat (A), the ulcer is larger in Tyrphostin-treated rat (B), reflecting delayed healing. The ulcer sizes in the Tyrphostin-treated rats (1.67 ± 0.11 mm) were significantly larger than those in vehicle-treated rats (0.89 ± 0.04 mm; P < 0.01). Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Changes in EGF-R levels in ulcer specimens of the vehicle- (■) and Tyrphostin- (□) treated rats. The detergent-solubilized immunoprecipitated samples of gastric specimens containing 150-μg proteins were subjected to Western immunoblot using specific polyclonal antibody against EGF-R. Density of protein bands was analyzed using a laser densitometer. Values represent relative densities of EGF-R levels (mean ± SEM; n = 6). *P < 0.02 vs. vehicle-treated group. n.s., nonsignificant. Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Tyrosine phosphorylation levels of EGF-R (170-kilodalton band) and other mucosal proteins in ulcer specimens from the vehicle- (■) and Tyrphostin- (□) treated rats were assessed by Western blot analysis. Tissue lysates containing 150-μg proteins were subjected to immunoblotting with antiphosphotyrosine antibody. Density of protein bands was analyzed using a laser densitometer. (A) Values represent relative density of phosphorylated EGF-R band (mean ± SEM; n = 6). *P < 0.05 vs. vehicle-treated group. (B) Representative Western blot showing tyrosine phosphorylation of EGF-R and other mucosal proteins. Lanes represent tyrosine phosphorylated proteins after the vehicle treatment (lane 1) and Tyrphostin treatment (lane 2) at 3 days and the vehicle treatment (lane 3) and Tyrphostin treatment (lane 4) at 7 days after ulcer induction. The Western blot is a representative of six separate experiments. Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 9 ERK1 activity in ulcer specimens from the vehicle- (■) and Tyrphostin- (□) treated rats. The activity was determined by measuring the levels of radiolabeled [γ-32P]ATP incorporated into MBP (pmol/mg protein) by ERK1 immunoprecipitated from cytosolic lysates. Values represent mean ± SEM of six experiments performed in triplicate. *P < 0.05 vs. vehicle-treated group. Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 10 ERK2 activity in ulcer specimens from the vehicle- (■) and Tyrphostin- (□) treated rats. The activity was determined by measuring the levels of radiolabeled [γ-32P]ATP incorporated into MBP (pmol/mg protein) by ERK2 immunoprecipitated from cytosolic lysates. Values represent mean ± SEM of six experiments performed in triplicate. *P < 0.03 vs. vehicle-treated group. Gastroenterology 1998 114, 706-713DOI: (10.1016/S0016-5085(98)70584-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions