Recombinant human BChE, human plasma, mouse plasma, rat plasma, cat plasma, mouse tissue extracts (liver, lungs, muscle) were used in the assay.
Hybridomas obtained during preparation were screened for antibody production by modified ELISA method and then by Western blot, immunohistochemistry, by ELISA with human, mouse, rat and cat plasma and mouse tissue extracts (liver, lungs, muscle).
Using synthetic peptides that contain methylated arginine residues, neither human PAD2, PAD3, PAD4, and PAD6 enzymes nor PADs present in mouse tissue extracts are capable of generating peptidyl citrulline or peptidyl methylcitrulline from either mono- or dimethylated peptidyl arginine.
Western blotting of mouse tissue extracts detected a 45 kD protein that was present most abundantly in the testis, and to a lesser degree in spleen, liver, lung and brain.
Furthermore, the nonspecific band detected in immunoblots appears to have some specificity for mammary glands since, to date, we have not detected this band in other mouse tissue extracts.
We determined NAT activity in cells or mouse tissue extracts by measuring the formation of N-acetylated metabolites using reverse-phase high-performance liquid chromatography (HPLC) as described previously (Grant et al. 1991).
Assays of GSK-3 were performed on mouse tissues extracts as described previously [12].
For comparison, glycomics analysis was conducted on mouse brain tissue extracts using glycoprotein immobilization for glycan extraction, which is described in detail previously.
To test the specificity of the antibodies, we conducted Western-blot analysis of mouse brain tissue extracts.
Correspondingly, KPNB1 strongly interacted with the endogenous PER2 in co-immunoprecipitation analysis using mouse liver tissue extracts.
The fact that Id-1 could be detected in mouse mammary tissue extracts by immunoblot analyses but was undetectable in mammary cells by immunohistochemical analyses led us to consider that the Id-1 detected in tissue extracts might have been derived from nonmammary cells.
