[Preparation and subcellular localization of antibody against RIP3].
OBJECTIVE
To determine the localization of RIP3 in NIH3T3 cell line and explore its roles in the signal transduction pathway of TNF-alpha-induced caspase-independent cell death.


METHODS
RIP3 gene was cloned from NIH3T3 cell line by RT-PCR and the amino acid 287-486 sequence of RIP3 was overexpressed in E.coli strain BL21. The protein was purified by electrodialysis to immune rabbits. Nine weeks later, the serum of the rabbits was harvested and anti-RIP3 polyclonal antibodies was purified and its specificity was detected by Western blot. Immunofluorescence was used to determine the subcellular localization of RIP3 in NIH3T3 cell line and the effects of TNF-alpha and z-VAD.fmk on RIP3 for its localization changing.


RESULTS
A high concentration of rabbit polyclonal antibody against RIP3 with good immunological characteristics was obtained. RIP3 was successfully detected by Western blot with anti-RIP3 antibody in 293T cells transfected with pcDNA3-flag-RIP3 and in mouse NIH3T3 cell line. The results of immunofluorescent staining displayed that RIP3 localized in cytoplasm in untreated NIH3T3 cells. Treatment with TNF-alpha or z-VAD. fmk alone didn't change its distribution. However in the cells induced by z-VAD.fmk and TNF-alpha, RIP3 was detected in both cytoplasm and nucleolus.


CONCLUSIONS
The polyclonal antibody against RIP3 with high concentration and specificity was successfully obtained. In NIH3T3 cell line, RIP3 localized in cytoplasm, which was subject to change induced by z-VAD.fmk and TNF-alpha, suggesting that RIP3 plays an important role in TNF-alpha induced caspase-independent cell death signal transduction pathway.
Xie
Lin-na
LN
School of Life Sciences, Xiamen University, Xiamen 361005, China.
Zhang
Na
N
Chen
Ming-liang
ML
Li
Qin-xi
QX
Zhou
Hua-min
HM
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism*; D000918:Antibody Specificity; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D000906:Antibodies; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse