PA5-52521

antibody from Invitrogen Antibodies

Targeting: NGEF ARHGEF27

Provider product page for PA5-52521

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [2]
Comments [0]
Validations
Immunohistochemistry [1]
Other assay [4]

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Product number: PA5-52521 - Provider product page
Provider: Invitrogen Antibodies
Product name: NGEF Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Recombinant full-length protein
Description: Immunogen sequence: METRESEDLE KTRRKSASDQ WNTDNEPAKV KPELLPEKEE TSQADQDIQD KEPHCHIPIK RNSIFNRSIR RKSKAKARDN PERNASCLAD SQDNGKSVNE PLTLNIPWSR TPPCRTAMQT DPGAQEMSES SST
Concentration: 0.1 mg/mL

NGEF protein structure - PA5-52521 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 1 Ephexin1 is upregulated in CRC and LC and promotes tumor cell growth and proliferation. A Expression of Ephexin1 mRNA in 12 primary cancer types from TCGA. Data are shown as mean +- SD. ns not significant, *** P < 0.001, two-tailed Student's t test. B Kaplan-Meier analysis of overall survival according to Ephexin1 in CRC and LC patients. P values are for a log-rank test. C Immunohistochemistry (IHC) staining was performed to evaluate Ephexin1 expression in CRC and LC tissues and their corresponding normal tissues. Hematoxylin is the counterstain. Scale bar = 100 mum. Data are shown as mean +- SD, P values are for a two-way ANOVA. D Ephexin1 protein expression in normal and indicated colon ( n = 8) and lung ( n = 11) cancer cell lines were examined by western blot. E Effect of Ephexin1 knockdown on cell proliferation by MTT assay in HCT116, H1299, H460, and A549 cells. Data are shown as mean +- SD. *** P < 0.001, two-way ANOVA. F , G Anchorage-independent growth assessed by soft agar assay ( F ) and migration ability ( G ) determined by transwell assay in Ephexin1-depleted HCT116, H1299, H460, and A549 cells. Representative images are shown. Scale bar = 100 mum. Data are shown as mean +- SD. ** P < 0.01, and *** P < 0.001, two-tailed Student's t test. H Control and Ephexin1-depleted HCT116, H1299, and H460 cells were inoculated subcutaneously in BALB/C nude mice ( n = 4). Tumor volumes recoded at the indicated times are shown. Data are shown as mean +- SD. *** P < 0.001

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 Ephexin1 enhances the MEK/ERK signaling pathway and regulates K-Ras-downstream target genes. A Whole-cell lysates from control or Ephexin1-depleted H1299 cells were incubated with human phosphokinase array membranes. Each pair of the most positive kinase dots is numbered with the identification of the corresponding kinase listed. B Levels of indicated protein kinases were quantified after normalized positive control. Error bars indicate mean +- SD of spots. C Western blot analysis to detect MEK/ERK activation in indicated cells treated with EGF for the indicated amounts of time after serum starvation. D The co-expression heat map of Ephexin1 with K-Ras, H-Ras, N-Ras, or Ki-67 in TCGA and GTEx ( n = 5151) derived from the UCSC Xena browser. E Heat map showing the differential expressed genes induced by Ephexin1 shRNA in H1299 cells. Red and green indicate high and low mRNA expression levels, respectively. F Scatter plot shows the differently expressed genes (FDR = 2) in Ephexin1-depleted H1299 cells compared with control cells. Compared to the control group (shControl), shEphexin1 downregulates 155 genes, whereas upregulates 227 genes expression levels. White circles highlight the position of the indicated K-Ras-downstream target genes. G Gene set enrichment analysis (GSEA) was used to identify K-Ras-downstream target genes differentially expressed between control and Ephexin1-depleted H1299 cells. H Heatmap of Ras-target genes downregulate

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5 Phosphorylation of Ephexin1 at Ser16/18 promotes tumor cell growth and proliferation. A Lysates from control and Ephexin1-depleted HEK293T cells transfected with V5-tagged C-Raf along with or without HA-tagged K-Ras G12V were immunoprecipitated with anti-V5 antibody and subjected to western blot analysis with indicated antibodies. B Co-IP analysis was conducted in HEK293T cells cotransfected with V5-C-Raf and Flag-Ephexin1 (WT, S16D/S18D, or S16A/S18A) with or without HA-K-Ras G12V . Cell lysates were immunoprecipitated with anti-V5 antibody and subjected to western blot analysis with indicated antibodies. C Co-IP analysis was conducted in HEK293T cells cotransfected with WT HA-K-Ras and Flag-Ephexin1 (WT, S16A/S18A, or S16D/S18D). Cell lysates were immunoprecipitated with anti-HA antibody and subjected to western blot analysis with indicated antibodies. D Co-IP analysis was conducted in HEK293T cells cotransfected with Flag-C-Raf and V5-Ephexin1 (WT, S16A/S18A, or S16D/S18D). Cell lysates were immunoprecipitated with anti-Flag antibody and subjected to western blot analysis with indicated antibodies. E Western blot analysis for Ephexin1 expression in indicated Ephexin1-depleted H1299 and HEK293T cells. F Anchorage-independent growth assessed by soft agar assay in Ephexin1-depleted H1299 cells transfected with Flag-tagged WT or mutant Ephexin1 (top) or in HEK293T cells transfected Flag-tagged WT or mutant Ephexin1 (bottom). Representative images are shown. Scale bar =

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 6 Ephexin1 is phosphorylated at Ser16/18 in human CRC and LC tissues and is associated with poor prognoses. A Representative image of pSer16/18 Ephexin1 immunoreactivity in normal and cancerous colorectal and lung tissues (separated by dashed lines). B , C IHC staining was performed to evaluate pSer16/18 Ephexin1 expression in normal, grade I/II, grade III/IV, and metastatic CRC ( B ) and LC ( C ) tissues and their corresponding normal tissues. Hematoxylin is the counterstain. Scale bar = 100 mum. Expression scores of pSer16/18 Ephexin1 in CRC tissue microarray (TMA) that was containing 9 cases of normal mucosa tissues, 14 cases grade I/II, 26 cases grade III/IV, and 10 cases of metastatic tumor tissues. Expression scores of pSer16/18 Ephexin1 in LC TMA that was containing 9 cases of normal mucosa tissues, 27 cases grade I/II, 13 cases grade III/IV, and 10 cases of metastatic tumor tissues. Data are shown as mean +- SD, P values are for a Mann-Whitney test. D A Kaplan-Meier graph representing overall survival rates for patients with pSer16/18 Ephexin1-overexpressing CRC. High pSer16/18 Ephexin1 expression, n = 33; low pSer16/18 Ephexin1 expression, n = 12. P values are for a log-rank test. E As in ( D ) but for LC. High pSer16/18 Ephexin1 expression, n = 42; low pSer16/18 Ephexin1 expression, n = 8. P values are for a log-rank test. F Schematic representation of a model for the major mechanism of the tumorigenic effect of Akt-mediated Ephexin1 phosphorylation on CRC and