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Production and characterization of ZFP36L1 antiserum against recombinant protein from Escherichia coli
Author(s) -
Cao Heping,
Lin Rui,
Ghosh Sanjukta,
Anderson Richard A.,
Urban Joseph F.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1003.7
Subject(s) - antiserum , polyclonal antibodies , recombinant dna , microbiology and biotechnology , messenger rna , tumor necrosis factor alpha , blot , escherichia coli , lipopolysaccharide , chemistry , cell culture , tristetraprolin , biology , antibody , gene , biochemistry , immunology , genetics , rna binding protein
Tristetraprolin (TTP/ZFP36) family proteins are anti‐inflammatory. They bind and destabilize some AU‐rich element‐containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was over‐expressed in E. coli, purified, and used for polyclonal antibody production in rabbits. This antiserum, and an antiserum against recombinant mouse TTP, was used to detect ZFP36L1 and TTP in mouse 3T3‐L1 adipocytes and RAW264.7 macrophages. Immunoblotting showed that ZFP36L1 was stably expressed, but TTP was induced by insulin and cinnamon and not by lipopolysaccharide (LPS) in 3T3‐L1 adipocytes. In contrast, ZFP36L1 was undetectable but TTP was strongly induced in LPS‐stimulated RAW264.7 cells. Quantitative real‐time polymerase chain reaction (PCR) confirmed higher levels of ZFP36L1 mRNA in adipocytes and TTP mRNA in RAW264.7 cells. Low levels of ZFP36L1 expression were also confirmed by northern blotting in mouse embryonic fibroblasts. These results demonstrate that TTP and ZFP36L1 are differentially expressed at the mRNA and protein levels in mouse adipocytes and macrophages (supported in part by the Intramural Research Program of the NIH, NIEHS and USDA‐ARS Human Nutrition Research Program).