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Western blot analyses of PGE2-related proteins and key signaling molecules in fibrosis.

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posted on 29.04.2015, 03:29 by Takeo Nakanishi, Yoshitaka Hasegawa, Reo Mimura, Tomohiko Wakayama, Yuka Uetoko, Hisakazu Komori, Shin-ichi Akanuma, Ken-ichi Hosoya, Ikumi Tamai

(A) Expression of proteins related to PGE2 metabolism and activation of signaling molecules were studied by Western blot analysis using lung homogenates prepared from WT and Slco2a1-/- mice (six per each group), and representative images are shown. (B) Quantitative analysis of each protein or phosphorylation was performed. Degree of expression of Cox2 and 15-Pgdh are shown by normalizing band intensity with that corresponding to Gapdh. Activation of phosphorylation of molecules was shown by normalizing band intensity for phosphorylated protein over the intensity for its total expression. Bands for phosphorylation of PKCδ and PKCθ were distinguished by molecular size on the blots. Phosphorylation of PKCα and βII was not distinguished by molecular size; therefore, we normalized their phosphorylation with total expression of PKCα and then the ratio was compared. Expression or degree of activation was compared between WT (open column) and Slco2a1-/- (closed column) mice, and each column represents the mean with SEM (lung tissues from 5 or 6 individual mice). Student’s t-test was used for statistical analysis (*, p < 0.05).