AH109 yeast cells were transformed with the following plasmids: 1, pAD-λcI plus pBD-λcI (positive control) 2, RS1 containing clone plus truncated IRIP bait 3, RS1 containing clone plus pBD-lamin C (negative control) 4, RS1 containing clone plus empty pBD vector (negative control) 5, RS1 containing clone plus pBD-λcI (negative control) and 6, pAD-λcI plus truncated IRIP bait (negative control). (A) Interaction of mIRIP and mRS1 in the yeast two-hybrid system. Interaction between mouse IRIP and mouse RS1. The lowest blot shows that there was no labeling in any of the samples after blocking antibodies with the purified GST-IRIP fusion protein. Cytosolic, nuclear, and plasma membrane fraction extracts were used for Western blot analysis with anti-mIRIP antibodies. (E) Intracellular distribution was tested in subcellular fractions from mouse kidney. The ∼30-kDa bands, visible only in samples immunoprecipitated with the antiserum, are indicated with arrows. ![]() The precipitates were resolved on 15% SDS-PAGE gel. Total cell extracts were prepared and immunoprecipitated with either preimmune or anti-mIRIP antibodies. Mouse NIH 3T3 cells were metabolically labeled with methionine and cysteine. (D) Immunoprecipitation of endogenous IRIP with polyclonal rabbit antibodies. After blocking, mIRIP was not detected its position is shown by an arrowhead. (C) Specificity of antibodies was also confirmed by testing cellular extracts from myc-tagged mIRIP (pEF/myc/mIRIP)-transfected cells with antibodies which were preadsorbed with GST-mIRIP. The recombinant protein was detected only in cells transfected with mIRIP. (B) Cellular extracts were prepared from HeLa and NIH 3T3 cells transiently transfected with myc-tagged mIRIP construct (+) or cells transfected with expression vector (−). The purified GST-mIRIP was detected by antibodies in Western blots. coli was used for generation of rabbit polyclonal antibodies. (A) Purified mIRIP protein expressed in E. Generation of anti-IRIP antibiotics and expression of IRIP in cell lines and kidney subcellular fractions. On the basis of these results, we propose that IRIP regulates the activity of a variety of transporters under normal and pathological conditions. We measured transport kinetics of OCT2-mediated uptake and demonstrated that IRIP overexpression significantly decreased V(max) but did not affect K(m). Conversely, inhibition of IRIP expression by small interfering RNA or antisense RNA increased MPP+ uptake. The activities of exogenous organic cation transporters (OCT2 and OCT3), organic anion transporter (OAT1), and monoamine transporters were also inhibited by IRIP. IRIP overexpression inhibited endogenous 1-methyl-4-phenylpyridinium (MPP+) uptake activity in HeLa cells. A possible role of IRIP in regulating transporter activity was subsequently investigated. The interaction between IRIP and RS1 was further confirmed in coimmunoprecipitation assays. ![]() The transporter regulator RS1 was identified as an IRIP-interacting protein in yeast two-hybrid screening. Besides ischemia/reperfusion, endotoxemia also activated the expression of IRIP in the liver, lung, and spleen. Mouse IRIP mRNA was expressed in all tissues tested, the highest level being in the testis, secretory, and endocrine organs. IRIP cDNA was isolated in a differential display analysis of an ischemia/reperfusion-treated kidney RNA sample. We report the identification and characterization of a new ischemia/reperfusion-inducible protein (IRIP), which belongs to the SUA5/YrdC/YciO protein family.
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