Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, R||IP, WB||Rb||Purified||Polyclonal Antibody|
|Presentation||0.1M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide before the addition of glycerol to 30%|
|Application||Detect RKIP using this Anti-RKIP Antibody validated for use in IP & WB.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||2 years at -20°C|
|Material Size||200 µg|
|Anti-RKIP (rabbit polyclonal IgG) - 2349629||2349629|
|Anti-RKIP (rabbit polyclonal IgG) - 2208815||2208815|
|Anti-RKIP - 2433435||2433435|
|Anti-RKIP - 20152||20152|
|Anti-RKIP - 2037641||2037641|
|Anti-RKIP - DAM1807838||DAM1807838|
|Anti-RKIP - JBC1852379||JBC1852379|
|Anti-RKIP - JBC1908440||JBC1908440|
|Reference overview||Application||Pub Med ID|
|Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells. |
Mulvey, HE; Chang, A; Adler, J; Del Tatto, M; Perez, K; Quesenberry, PJ; Chatterjee, D
BMC cancer 15 571 2015
Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype.EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB.This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.
|Raf kinase inhibitor protein (RKIP) blocks signal transducer and activator of transcription 3 (STAT3) activation in breast and prostate cancer. |
Yousuf, S; Duan, M; Moen, EL; Cross-Knorr, S; Brilliant, K; Bonavida, B; LaValle, T; Yeung, KC; Al-Mulla, F; Chin, E; Chatterjee, D
PloS one 9 e92478 2014
Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding-protein (PEBP) family that modulates the action of many kinases involved in cellular growth, apoptosis, epithelial to mesenchymal transition, motility, invasion and metastasis. Previously, we described an inverse association between RKIP and signal transducers and activators of transcription 3 (STAT3) expression in gastric adenocarcinoma patients. In this study, we elucidated the mechanism by which RKIP regulates STAT3 activity in breast and prostate cancer cell lines. RKIP over expression inhibited c-Src auto-phosphorylation and activation, as well as IL-6-, JAK1 and 2-, and activated Raf-mediated STAT3 tyrosine and serine phosphorylation and subsequent activation. In MDA-231 breast cancer cells that stably over express RKIP, IL-6 treatment blocked STAT3 phosphorylation and transcriptional activation. Conversely, in RKIP knockdown MDA-231 cells: STAT3 phosphorylation and activation increased in comparison to parental MDA-231 cells. RKIP over expression resulted in constitutive physical interaction with STAT3 and blocked c-Src and STAT3 association. The treatment of DU145 prostate, but not PC3 prostate or MDA-231 breast, cancer cell lines with ENMD-1198 or MKC-1 dramatically increased expression of RKIP. Overexpression of RKIP sensitized PC3 and MDA-231 cells to MTI-induced apoptosis. Moreover, MTI treatment resulted in a decrease in Src-mediated STAT3 tyrosine phosphorylation and activation, an effect that was significantly enhanced by RKIP over expression. In stable RKIP over expressing MDA-231 cells, tumor xenograft growth induced by activated STAT3 is inhibited. RKIP synergizes with MTIs to induce apoptosis and inhibit STAT3 activation of breast and prostate cancer cells. RKIP plays a critical role in opposing the effects of pro-oncogenic STAT3 activation.
|Geographic analysis of RKIP expression and its clinical relevance in colorectal cancer. |
Koelzer, VH; Karamitopoulou, E; Dawson, H; Kondi-Pafiti, A; Zlobec, I; Lugli, A
British journal of cancer 108 2088-96 2013
This study evaluates the geographic expression pattern of Raf-1 Kinase Inhibitor Protein (RKIP) in colorectal cancer (CRC) in correlation with clinicopathological and molecular features, markers of epithelial-mesenchymal transition (EMT) and survival outcome.Whole-tissue sections of 220 well-characterised CRCs were immunostained for RKIP. NF-κB and E-Cadherin expression was assessed using a matched multi-punch tissue microarray. Analysis of mismatch repair (MMR) protein expression, B-Raf and KRAS mutations was performed. RKIP expression in normal mucosa, tumour centre, invasion front and tumour buds was each assessed for clinical relevance.RKIP was diffusely expressed in normal mucosa and progressively lost towards tumour centre and front (Pless than 0.0001). Only 0.9% of tumour buds were RKIP-positive. In the tumour centre, RKIP deficiency predicted metastatic disease (P=0.0307), vascular invasion (P=0.0506), tumour budding (P=0.0112) and an invasive border configuration (P=0.0084). Loss of RKIP correlated with NF-κB activation (P=0.0002) and loss of E-Cadherin (Pless than 0.0001). Absence of RKIP was more common in MMR-deficient cancers (P=0.0191), while no impact of KRAS and B-Raf mutation was observed. RKIP in the tumour centre was identified as a strong prognostic indicator (HR (95% CI): 2.13 (1.27-3.56); P=0.0042) independently of TNM classification and therapy (P=0.0474).The clinical relevance of RKIP expression as an independent prognostic factor is restricted to the tumour centre. Loss of RKIP predicts features of EMT and correlates with frequent distant metastasis.
|RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients. |
Cross-Knorr, S; Lu, S; Perez, K; Guevara, S; Brilliant, K; Pisano, C; Quesenberry, PJ; Resnick, MB; Chatterjee, D
BMC cancer 13 463 2013
A major obstacle in treating colorectal cancer (CRC) is the acquired resistance to chemotherapeutic agents. An important protein in the regulation of cancer cell death and clinical outcome is Raf kinase inhibitor protein (RKIP). In contrast, activated signal transducer and activator of transcription 3 (STAT3) is a protein that promotes tumor cell survival by inhibiting apoptosis and has an important role in cancer progression in many of cancer types. The aim of this study was to evaluate the regulation of RKIP and STAT3 after treatment with clinically relevant chemotherapeutic agents (camptothecin (CPT) and oxaliplatin (OXP)) and the cytokine interleukin-6 (IL-6) in HCT116 colon cancer cells as well as evaluate the association between RKIP and STAT3 with clinical outcome of Stage II colon cancer patients.HCT-116 colon cancer cells were treated with CPT, OXP, and IL-6 separately or in combination in a time and dose-dependent manner and examined for phosphorylated and non-phosphorylated RKIP and STAT3 via Western blot analysis. STAT3 transcriptional activity was measured via a luciferase reporter assay in HCT116 cells treated with CPT, IL-6 or transfected with JAK 1, 2 separately or in combination. We extended these observations and determined STAT3 and RKIP/ pRKIP in tumor microarrays (TMA) in stage II colon cancer patients.We demonstrate IL-6-mediated activation of STAT3 occurs in conjunction with the phosphorylation of RKIP in vitro in human colon cancer cells. OXP and CPT block IL-6 mediated STAT3 activation and RKIP phosphorylation via the inhibition of the interaction of STAT3 with gp130. We determined that STAT3 and nuclear pRKIP are significantly associated with poor patient prognosis in stage II colon cancer patients.In the analysis of tumor samples from stage II colon cancer patients and the human colon carcinoma cell line HCT116, pRKIP and STAT3, 2 proteins potentially involved in the resistance to conventional treatments were detected. The phosphorylation of pRKIP and STAT3 are induced by the cytokine IL-6 and suppressed by the chemotherapeutic drugs CPT and OXP. Therefore, these results suggest that STAT3 and pRKIP may serve as prognostic biomarkers in stage II colon cancer patients and may improve chemotherapy.
|RKIP inhibition in cervical cancer is associated with higher tumor aggressive behavior and resistance to cisplatin therapy. |
Martinho, O; Pinto, F; Granja, S; Miranda-Gonçalves, V; Moreira, MA; Ribeiro, LF; di Loreto, C; Rosner, MR; Longatto-Filho, A; Reis, RM
PloS one 8 e59104 2013
Cervical cancer is one of the most common cancers in women worldwide, being high-risk group the HPV infected, the leading etiological factor. The raf kinase inhibitory protein (RKIP) has been associated with tumor progression and metastasis in several human neoplasms, however its role on cervical cancer is unclear. In the present study, 259 uterine cervix tissues, including cervicitis, cervical intraepithelial lesions and carcinomas, were analyzed for RKIP expression by immunohistochemistry. We found that RKIP expression was significantly decreased during malignant progression, being highly expressed in non-neoplastic tissues (54% of the samples; 73/135), and expressed at low levels in the cervix invasive carcinomas (∼15% (19/124). Following in vitro downregulation of RKIP, we observed a viability and proliferative advantage of RKIP-inhibited cells over time, which was associated with an altered cell cycle distribution and higher colony number in a colony formation assay. An in vitro wound healing assay showed that RKIP abrogation is associated with increased migratory capability. RKIP downregulation was also associated with an increased vascularization of the tumors in vivo using a CAM assay. Furthermore, RKIP inhibition induced cervical cancer cells apoptotic resistance to cisplatin treatment. In conclusion, we described that RKIP protein is significantly depleted during the malignant progression of cervical tumors. Despite the lack of association with patient clinical outcome, we demonstrate, in vitro and in vivo, that loss of RKIP expression can be one of the factors that are behind the aggressiveness, malignant progression and chemotherapy resistance of cervical cancer.
|Novel proteins associated with human dilated cardiomyopathy: selective reduction in α(1A)-adrenergic receptors and increased desensitization proteins. |
Shi, T; Moravec, CS; Perez, DM
Journal of receptor and signal transduction research 33 96-106 2013
Abstract Therapeutics to treat human heart failure (HF) and the identification of proteins associated with HF are still limited. We analyzed α(1)-adrenergic receptor (AR) subtypes in human HF and performed proteomic analysis on more uniform samples to identify novel proteins associated with human HF. Six failing hearts with end-stage dilated cardiomyopathy (DCM) and four non-failing heart controls were subjected to proteomic analysis. Out of 48 identified proteins, 26 proteins were redundant between samples. Ten of these 26 proteins were previously reported to be associated with HF. Of the newly identified proteins, we found several muscle proteins and mitochondrial/electron transport proteins, while novel were functionally similar to previous reports. However, we also found novel proteins involved in functional classes such as β-oxidation and G-protein coupled receptor signaling and desensitization not previously associated with HF. We also performed radioligand-binding studies on the heart samples and not only confirmed a large loss of β(1)-ARs in end-stage DCM, but also found a selective decrease in the α(1A)-AR subtype not previously reported. We have identified new proteins and functional categories associated with end-stage DCM. We also report that similar to the previously characterized loss of β(1)-AR in HF, there is also a concomitant loss of α(1A)-ARs, which are considered cardioprotective proteins.
|Proteomic changes in rat spermatogenesis in response to in vivo androgen manipulation; impact on meiotic cells. |
Stanton, PG; Sluka, P; Foo, CF; Stephens, AN; Smith, AI; McLachlan, RI; O'Donnell, L
PloS one 7 e41718 2012
The production of mature sperm is reliant on androgen action within the testis, and it is well established that androgens act on receptors within the somatic Sertoli cells to stimulate male germ cell development. Mice lacking Sertoli cell androgen receptors (AR) show late meiotic germ cell arrest, suggesting Sertoli cells transduce the androgenic stimulus co-ordinating this essential step in spermatogenesis. This study aimed to identify germ cell proteins responsive to changes in testicular androgen levels and thereby elucidate mechanisms by which androgens regulate meiosis. Testicular androgen levels were suppressed for 9 weeks using testosterone and estradiol-filled silastic implants, followed by a short period of either further androgen suppression (via an AR antagonist) or the restoration of intratesticular testosterone levels. Comparative proteomics were performed on protein extracts from enriched meiotic cell preparations from adult rats undergoing androgen deprivation and replacement in vivo. Loss of androgenic stimulus caused changes in proteins with known roles in meiosis (including Nasp and Hsp70-2), apoptosis (including Diablo), cell signalling (including 14-3-3 isoforms), oxidative stress, DNA repair, and RNA processing. Immunostaining for oxidised DNA adducts confirmed spermatocytes undergo oxidative stress-induced DNA damage during androgen suppression. An increase in PCNA and an associated ubiquitin-conjugating enzyme (Ubc13) suggested a role for PCNA-mediated regulation of DNA repair pathways in spermatocytes. Changes in cytoplasmic SUMO1 localisation in spermatocytes were paralleled by changes in the levels of free SUMO1 and of a subunit of its activating complex, suggesting sumoylation in spermatocytes is modified by androgen action on Sertoli cells. We conclude that Sertoli cells, in response to androgens, modulate protein translation and post-translational events in spermatocytes that impact on their metabolism, survival, and completion of meiosis.
|Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein. |
Yeung, K, et al.
Mol. Cell. Biol., 20: 3079-85 (2000) 2000
We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1-MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.
|Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. |
Yeung, K, et al.
Nature, 401: 173-7 (1999) 1999
Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK). This kinase cascade controls the proliferation and differentiation of different cell types. Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen. This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein). In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras. RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy. RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases. RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase. Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription. RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK module.