Figure 3. Alterations of PCNA and MAPK signal pathways in transplanted submandibular glands. Immunohistochemical localizations of PCNA in control submandibular gland (A), transplanted gland (B), and phenylephrine-treated gland (C) are shown. PCNA-positive cell is indicated by an arrow. Light microscopy, magnification 400x. Bars (A,B,C): 20 µm. (D) Semiquantitative scoring of PCNA-positive cells (n = 6). The PCNA-positive cells were counted in 10 different fields in each section (1 section/each animal) under 400x magnification. (E) Western blot analysis of phospho-PKC and PKC. An 80-µg quantity of protein was separated by 12% SDS-polyacrylamide gel electrophoresis and immunoblotted either with an antibody specific for phospho-PKC or for total PKC. (F) Western blot analysis of phospho-ERK1/2 and ERK1/2. An 80-µg quantity of protein was separated by 12% SDS-polyacrylamide gel electrophoresis and immunoblotted either with an antibody specific for phospho-ERK1/2 or for total ERK1/2. Filters were re-probed with antibody against actin to ensure equal loading of the lanes. The blot is a representative of 5 separated animals in each group, with similar results. All values are given as means ± SEM. N, control submandibular gland; T, transplanted gland; T + PE, transplanted gland with phenylephrine treatment. *P < 0.05 and **P < 0.01 compared with N; ^#P < 0.05 and ^##P < 0.01 compared with T.