Production and characterization of ZFP36L1 antiserum against recombinant protein from Escherichia coli.
Tristetraprolin/zinc finger protein 36 (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 overexpressed in E. coli, purified, and used for polyclonal antibody production in rabbits. The antiserum recognized nanograms of the antigen on immunoblots. This antiserum and another antiserum developed against recombinant mouse TTP were used to detect ZFP36L1 and TTP in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. Immunoblotting showed that ZFP36L1 was stably expressed with a size corresponding to the lower mass size of ZFP36L1 expressed in transfected human embryonic kidney 293 cells, but TTP was induced by cinnamon extract and not by lipopolysaccharide (LPS) in adipocytes. In contrast, ZFP36L1 was undetectable, but TTP was strongly induced in LPS-stimulated RAW cells. Quantitative real-time polymerase chain reaction confirmed the higher levels of ZFP36L1 mRNA in adipocytes and TTP mRNA in RAW cells. Low levels of ZFP36L1 expression were also confirmed by Northern blotting in mouse embryonic fibroblasts. These results demonstrate that ZFP36L1 antiserum is useful in the detection of this protein and that TTP and ZFP36L1 are differentially expressed and regulated at the mRNA and protein levels in mouse adipocytes and macrophages.
Cao
Heping
H
Diet, Genomics and Immunology Laboratory, Beltsville Human Nutrition Research Center, U.S. Department of Agriculture-ARS, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. heping.cao@ars.usda.gov
Lin
Rui
R
Ghosh
Sanjukta
S
Anderson
Richard A
RA
Urban
Joseph F
JF
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D051817:Butyrate Response Factor 1; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015151:Immunoblotting; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051816:Tristetraprolin / Q000096:biosynthesis / Q000235:genetics
D000906:Antibodies; D051817:Butyrate Response Factor 1; D017931:DNA Primers; D018076:DNA, Complementary; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D051816:Tristetraprolin; C488431:Zfp36l1 protein, mouse