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ABclonal Biotechnology ihc ikbke

Ihc Ikbke, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ikk%CE%B5+rabbit+pab+(a0244)/IKK%CE%B5+Rabbit+pAb/pm39664571-102-6-8
Average 92 stars, based on 3 article reviews

ihc ikbke - by Bioz Stars, 2026-10

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1) Product Images from "IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway."

Article Title: IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway.

Journal: International journal of biological sciences

doi: 10.7150/ijbs.102666

Figure Legend Snippet: Figure 1. IKBKE expression is upregulated and associated with poor survival in RCC. (A) The mRNA expression profile of IKBKE across several cancer types. (B) IKBKE mRNA expression in RCC and normal tissues from a TCGA dataset. (C) IKBKE mRNA expression in RCC and paired paracancerous tissues from a TCGA dataset. (D-E) IKBKE mRNA expression in RCC and normal tissues from GSE36985 and GSE16449. (F) Representative immunohistochemical staining of IKBKE in 60 paired RCC samples and normal samples (n = 60). Scale bars, 100 μm. (G) The IHC scores of IKBKE in normal tissues and RCC tissues (n = 60). (H-K) Correlations between IKBKE mRNA expression of RCC patients with clinical staging, including T, M, N, and grade. (L) The Kaplan–Meier plot of overall survival and progression free-survival for RCC grouped by IKBKE expression. (M) Multivariate Cox regression analysis adjusted for IKBKE expression, age, sex, grade, and stage. (N) The protein expression levels of IKBKE in RCC and normal kidney cell lines by Western blotting. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using the Mann–Whitney U test for (A, B, D, E, and H-K), Student’s t test for (C and G), and log‐rank test for (L). *p < 0.05, **p < 0.01, ***p < 0.001.

Techniques Used: Expressing, Immunohistochemical staining, Staining, Western Blot, MANN-WHITNEY

Figure 2. Oncogenic role of IKBKE in RCC. (A) Evaluation of IKBKE knockdown efficiency in RCC cell lines through Western blotting. (B) The cell proliferation rates in the shControl and shIKBKE groups were assessed in RCC cell lines through a CCK-8 assay. (C-D) The colony numbers in the shControl and shIKBKE groups were compared in RCC cell lines through a colony formation assay. The diameter of each well in panel C is approximately 35 mm. (E) Correlation between IKBKE mRNA expression and Cyclin B1 mRNA expression. (F-G) Cell cycle analysis of RCC cell lines with shControl or shIKBKE through flow cytometry. (H-I) The cell apoptosis of RCC cell lines with shControl or shIKBKE through flow cytometry. (J-L) 786-O cells stably expressing shControl or shIKBKE-specific shRNAs were injected subcutaneously into nude mice for xenograft assays. The xenografts in each group (n = 6 mice per group) were photographed in Panel J, the tumor mass is shown in Panel K, and the tumor growth curve is shown in Panel L. Each bar represents the mean values ± SD of three independent experiments except where stated. Statistical analysis was performed using one-way ANOVA for (B, D, and I), Pearson’s correlation analysis for (E), and Student’s t test for (K and L). ns, no significance, **p < 0.01, ***p < 0.001.

Figure Legend Snippet: Figure 2. Oncogenic role of IKBKE in RCC. (A) Evaluation of IKBKE knockdown efficiency in RCC cell lines through Western blotting. (B) The cell proliferation rates in the shControl and shIKBKE groups were assessed in RCC cell lines through a CCK-8 assay. (C-D) The colony numbers in the shControl and shIKBKE groups were compared in RCC cell lines through a colony formation assay. The diameter of each well in panel C is approximately 35 mm. (E) Correlation between IKBKE mRNA expression and Cyclin B1 mRNA expression. (F-G) Cell cycle analysis of RCC cell lines with shControl or shIKBKE through flow cytometry. (H-I) The cell apoptosis of RCC cell lines with shControl or shIKBKE through flow cytometry. (J-L) 786-O cells stably expressing shControl or shIKBKE-specific shRNAs were injected subcutaneously into nude mice for xenograft assays. The xenografts in each group (n = 6 mice per group) were photographed in Panel J, the tumor mass is shown in Panel K, and the tumor growth curve is shown in Panel L. Each bar represents the mean values ± SD of three independent experiments except where stated. Statistical analysis was performed using one-way ANOVA for (B, D, and I), Pearson’s correlation analysis for (E), and Student’s t test for (K and L). ns, no significance, **p < 0.01, ***p < 0.001.

Techniques Used: Knockdown, Western Blot, CCK-8 Assay, Colony Assay, Expressing, Cell Cycle Assay, Flow Cytometry, Stable Transfection, Injection

Figure 3. Correlation between IKBKE and sunitinib resistance in RCC. (A) The PI3K–AKT upregulated gene set was used to perform GSEA for the respective data sets with the IKBKE transcription level from TCGA and GSE40435 datasets. (B) The EGFR–Tyrosine-Kinase-Inhibitor-Resistance upregulated gene set was used to perform GSEA for the respective data sets with the IKBKE transcription level from GSE53757 and GSE68417 datasets. (C) IKBKE mRNA expression in sunitinib-escape and paired sunitinib-responsive samples from the GSE76068 dataset. IKBKE mRNA expression in sorafenib-resistant and untreated samples from the GSE64052 dataset. (D) Heatmap analysis showed the top 55 genes involved in sunitinib escape and response in the GSE76068 dataset. (E) PPI networks of IKBKE, RRM2, and 55 genes involved in sunitinib escape and response. (F) RRM2 and IKBKE protein expression levels were measured in 769-P and 786-O cells with shControl or shIKBKE by Western blotting. (G) RRM2 mRNA expression levels were measured in 769-P and 786-O cells with shControl or shIKBKE by qPCR. (H) The correlation between RRM2 expression and IKBKE expression for the data from TCGA database. (I) Representative immunohistochemical staining of RRM2 and IKBKE using the tissue microarray of renal cancer. Scale bars, 100 μm. (J) The correlation between IHC scores of RRM2 and IKBKE (n = 60). Each bar represents the mean values ± SD of three independent experiments except where stated. Statistical analysis was performed using Student’s t test for (C), one-way ANOVA for (G), and Pearson’s correlation analysis for (H and I). ns, no significance, *p < 0.05.

Figure Legend Snippet: Figure 3. Correlation between IKBKE and sunitinib resistance in RCC. (A) The PI3K–AKT upregulated gene set was used to perform GSEA for the respective data sets with the IKBKE transcription level from TCGA and GSE40435 datasets. (B) The EGFR–Tyrosine-Kinase-Inhibitor-Resistance upregulated gene set was used to perform GSEA for the respective data sets with the IKBKE transcription level from GSE53757 and GSE68417 datasets. (C) IKBKE mRNA expression in sunitinib-escape and paired sunitinib-responsive samples from the GSE76068 dataset. IKBKE mRNA expression in sorafenib-resistant and untreated samples from the GSE64052 dataset. (D) Heatmap analysis showed the top 55 genes involved in sunitinib escape and response in the GSE76068 dataset. (E) PPI networks of IKBKE, RRM2, and 55 genes involved in sunitinib escape and response. (F) RRM2 and IKBKE protein expression levels were measured in 769-P and 786-O cells with shControl or shIKBKE by Western blotting. (G) RRM2 mRNA expression levels were measured in 769-P and 786-O cells with shControl or shIKBKE by qPCR. (H) The correlation between RRM2 expression and IKBKE expression for the data from TCGA database. (I) Representative immunohistochemical staining of RRM2 and IKBKE using the tissue microarray of renal cancer. Scale bars, 100 μm. (J) The correlation between IHC scores of RRM2 and IKBKE (n = 60). Each bar represents the mean values ± SD of three independent experiments except where stated. Statistical analysis was performed using Student’s t test for (C), one-way ANOVA for (G), and Pearson’s correlation analysis for (H and I). ns, no significance, *p < 0.05.

Techniques Used: Expressing, Western Blot, Immunohistochemical staining, Staining, Microarray

Figure 4. IKBKE binds to RRM2 to activate AKT in RCC. (A) The expression of CyclinB1 and CyclinA was determined by Western blotting in RCC cell lines treated with shIKBKE. (B) The expression of RRM2 was determined by Western blotting in RCC cell lines treated with shIKBKE or MG132. (C) Indicated plasmids were transfected into HEK293T cells and an anti-Flag antibody was used for immunoprecipitation. The precipitates were immunoblotted using Myc- and anti-Flag antibodies. (D) The whole-cell lysates of 769-P and 786-O cells were precipitated with IKBKE antibodies, and the precipitates were examined by immunoblotting to evaluate the endogenous interaction between IKBKE and RRM2. (E) RRM2-Flags were co-transfected with IKBKE plasmids into HEK293T cells, and the whole-cell lysates were subjected to immuno-affinity purification using anti-Flag magnetic beads. The expression of phospho-serine/threonine/tyrosine (p/S/T/Y) was evaluated by Western blotting. (F) 786-O cells were infected with the indicated lentivirus for 72 h, and subsequently, the cells were harvested for Western blotting to determine the expression of target proteins. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using Student’s t test for (E and F). ns, no significance, *p < 0.05, **p < 0.01.

Figure Legend Snippet: Figure 4. IKBKE binds to RRM2 to activate AKT in RCC. (A) The expression of CyclinB1 and CyclinA was determined by Western blotting in RCC cell lines treated with shIKBKE. (B) The expression of RRM2 was determined by Western blotting in RCC cell lines treated with shIKBKE or MG132. (C) Indicated plasmids were transfected into HEK293T cells and an anti-Flag antibody was used for immunoprecipitation. The precipitates were immunoblotted using Myc- and anti-Flag antibodies. (D) The whole-cell lysates of 769-P and 786-O cells were precipitated with IKBKE antibodies, and the precipitates were examined by immunoblotting to evaluate the endogenous interaction between IKBKE and RRM2. (E) RRM2-Flags were co-transfected with IKBKE plasmids into HEK293T cells, and the whole-cell lysates were subjected to immuno-affinity purification using anti-Flag magnetic beads. The expression of phospho-serine/threonine/tyrosine (p/S/T/Y) was evaluated by Western blotting. (F) 786-O cells were infected with the indicated lentivirus for 72 h, and subsequently, the cells were harvested for Western blotting to determine the expression of target proteins. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using Student’s t test for (E and F). ns, no significance, *p < 0.05, **p < 0.01.

Techniques Used: Expressing, Western Blot, Transfection, Immunoprecipitation, Affinity Purification, Magnetic Beads, Infection

Figure 5. IKBKE promotes RCC progression by upregulating RRM2. (A) The expression of IKBKE and RRM2 was measured in RCC cell lines treated with shIKBKE or shRRM2 by Western blotting. (B) The cell proliferation rate of RCC cell lines infected with the indicated lentivirus was evaluated through CCK-8 assays. (C) The expression of IKBKE and RRM2 was measured in RCC cell lines treated with IKBKE or shRRM2 by Western blotting. (D) The cell proliferation rate of RCC cell lines infected with the indicated lentivirus was evaluated through CCK-8 assays. (E-H) The colony numbers of RCC cell lines infected with the indicated lentivirus were evaluated through colony formation assays. The diameter of each well in panel C is approximately 35 mm. (I-J) The cell apoptosis of RCC cell lines infected with the indicated lentivirus was evaluated by Annexin V-7AAD flow cytometry. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA for (B, D, F, and H). ns, no, significance, **p < 0.01, ***p < 0.001.

Figure Legend Snippet: Figure 5. IKBKE promotes RCC progression by upregulating RRM2. (A) The expression of IKBKE and RRM2 was measured in RCC cell lines treated with shIKBKE or shRRM2 by Western blotting. (B) The cell proliferation rate of RCC cell lines infected with the indicated lentivirus was evaluated through CCK-8 assays. (C) The expression of IKBKE and RRM2 was measured in RCC cell lines treated with IKBKE or shRRM2 by Western blotting. (D) The cell proliferation rate of RCC cell lines infected with the indicated lentivirus was evaluated through CCK-8 assays. (E-H) The colony numbers of RCC cell lines infected with the indicated lentivirus were evaluated through colony formation assays. The diameter of each well in panel C is approximately 35 mm. (I-J) The cell apoptosis of RCC cell lines infected with the indicated lentivirus was evaluated by Annexin V-7AAD flow cytometry. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA for (B, D, F, and H). ns, no, significance, **p < 0.01, ***p < 0.001.

Techniques Used: Expressing, Western Blot, Infection, CCK-8 Assay, Flow Cytometry

Figure 6. The IKBKE–RRM2 axis regulates sunitinib sensitivity in RCC. (A-H) 786-O cells were infected with the indicated lentivirus for 72 h, and subsequently, the cells were treated with or without sunitinib (2 µM). The cell proliferation rate was evaluated through CCK-8 (A, C) and EDU (E and G, scale bars, 50 μm) assays, and cell apoptosis was evaluated by Annexin V-7AAD flow cytometry (B, D). (I-J) 769-P and 786-O cells were infected with the indicated lentivirus for 72 h, and subsequently, the cells were treated with a series of concentrations of sunitinib for 48 h. Cell viability was determined by a CCK-8 assay. (K-M) 786-O cells were infected with the indicated lentivirus for 72 h. After puromycin (5 μg/mL) selection, cells were collected and subcutaneously injected into the nude mice (n = 6 mice per group). These mice were treated with or without sunitinib (20 mg/kg). The xenografts in each group are shown in Panel K, the tumor mass is shown in Panel L, and the tumor growth curve is shown in Panel M. Each bar represents the mean values ± SD of three independent experiments except where stated. Statistical analysis was performed using one-way ANOVA for (A, C, F, H, L, and M). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure Legend Snippet: Figure 6. The IKBKE–RRM2 axis regulates sunitinib sensitivity in RCC. (A-H) 786-O cells were infected with the indicated lentivirus for 72 h, and subsequently, the cells were treated with or without sunitinib (2 µM). The cell proliferation rate was evaluated through CCK-8 (A, C) and EDU (E and G, scale bars, 50 μm) assays, and cell apoptosis was evaluated by Annexin V-7AAD flow cytometry (B, D). (I-J) 769-P and 786-O cells were infected with the indicated lentivirus for 72 h, and subsequently, the cells were treated with a series of concentrations of sunitinib for 48 h. Cell viability was determined by a CCK-8 assay. (K-M) 786-O cells were infected with the indicated lentivirus for 72 h. After puromycin (5 μg/mL) selection, cells were collected and subcutaneously injected into the nude mice (n = 6 mice per group). These mice were treated with or without sunitinib (20 mg/kg). The xenografts in each group are shown in Panel K, the tumor mass is shown in Panel L, and the tumor growth curve is shown in Panel M. Each bar represents the mean values ± SD of three independent experiments except where stated. Statistical analysis was performed using one-way ANOVA for (A, C, F, H, L, and M). *p < 0.05, **p < 0.01, ***p < 0.001.

Techniques Used: Infection, CCK-8 Assay, Flow Cytometry, Selection, Injection

Figure 7. The IKBKE inhibitor CYT387 regulates sunitinib sensitivity in RCC. (A) 769-P and 786-O cells were treated with or without 5 μM CYT387 for 48 h. The protein expression of IKBKE and RRM2 was detected by Western blotting. (B) 769-P and 786-O cells were treated with control, DMSO or CYT387 (5 μM) for 48 h; subsequently, these cells were harvested and treated with a series of doses of sunitinib for 48 h. Cell viability was determined by CCK-8 assays. (C-F) 786-O cells were treated with DMSO or CYT387 for 48 h. Subsequently, these cells were treated with sunitinib (2 µM). Cell apoptosis was evaluated by Annexin V-7AAD flow cytometry (C, D). The cell proliferation rate was evaluated by EDU assays (E, F). Scale bar = 50 μm. (G-H) 786-O cells were treated with indicated constructs. After 48 h, the cell medium was collected for in vitro angiogenesis assays. Scale bar = 100 μm. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA for (D, F, and H). **p < 0.01, ***p < 0.001.

Figure Legend Snippet: Figure 7. The IKBKE inhibitor CYT387 regulates sunitinib sensitivity in RCC. (A) 769-P and 786-O cells were treated with or without 5 μM CYT387 for 48 h. The protein expression of IKBKE and RRM2 was detected by Western blotting. (B) 769-P and 786-O cells were treated with control, DMSO or CYT387 (5 μM) for 48 h; subsequently, these cells were harvested and treated with a series of doses of sunitinib for 48 h. Cell viability was determined by CCK-8 assays. (C-F) 786-O cells were treated with DMSO or CYT387 for 48 h. Subsequently, these cells were treated with sunitinib (2 µM). Cell apoptosis was evaluated by Annexin V-7AAD flow cytometry (C, D). The cell proliferation rate was evaluated by EDU assays (E, F). Scale bar = 50 μm. (G-H) 786-O cells were treated with indicated constructs. After 48 h, the cell medium was collected for in vitro angiogenesis assays. Scale bar = 100 μm. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA for (D, F, and H). **p < 0.01, ***p < 0.001.

Techniques Used: Expressing, Western Blot, Control, CCK-8 Assay, Flow Cytometry, Construct, In Vitro

Figure 8. A working model of the IKBKE–RRM2 axis regulating RCC progression and sunitinib resistance. (Left) IKBKE overexpression induces RCC progression and sunitinib resistance by upregulating RRM2 through the RRM2-mediated PI3K/AKT pathway. (Right) IKBKE silencing downregulates RRM2 and induces cell cycle arrest at G2/M phase through the RRM2-mediated PI3K/AKT pathway, suppressing the progression of renal cancer cells and enhancing sunitinib sensitivity. In addition, the IKBKE inhibitor CYT387 can inhibit IKBKE expression and restore sunitinib sensitivity in RCC cells.

Figure Legend Snippet: Figure 8. A working model of the IKBKE–RRM2 axis regulating RCC progression and sunitinib resistance. (Left) IKBKE overexpression induces RCC progression and sunitinib resistance by upregulating RRM2 through the RRM2-mediated PI3K/AKT pathway. (Right) IKBKE silencing downregulates RRM2 and induces cell cycle arrest at G2/M phase through the RRM2-mediated PI3K/AKT pathway, suppressing the progression of renal cancer cells and enhancing sunitinib sensitivity. In addition, the IKBKE inhibitor CYT387 can inhibit IKBKE expression and restore sunitinib sensitivity in RCC cells.

Techniques Used: Over Expression, Expressing

other:

Article Title: SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy.
Article Snippet: SERPINB1 Rabbit pAb (A6257), STAT1 Rabbit pAb (A12075), Phospho-STAT1Y701 Rabbit mAb (AP0054), and Ikkε Rabbit pAb (A0244) were purchased from ABclonal. mTOR (7C10) Rabbit mAb (#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (#5536), AKT (pan) (C67E7) Rabbit mAb (#4691) and Phospho-AKT (Ser473) (D9E) XP Rabbit mAb (#4060) were purchased from the Cell Signaling Technology.

Image Search Results

Journal: International journal of biological sciences

Article Title: IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway.

doi: 10.7150/ijbs.102666

Figure Lengend Snippet: Figure 1. IKBKE expression is upregulated and associated with poor survival in RCC. (A) The mRNA expression profile of IKBKE across several cancer types. (B) IKBKE mRNA expression in RCC and normal tissues from a TCGA dataset. (C) IKBKE mRNA expression in RCC and paired paracancerous tissues from a TCGA dataset. (D-E) IKBKE mRNA expression in RCC and normal tissues from GSE36985 and GSE16449. (F) Representative immunohistochemical staining of IKBKE in 60 paired RCC samples and normal samples (n = 60). Scale bars, 100 μm. (G) The IHC scores of IKBKE in normal tissues and RCC tissues (n = 60). (H-K) Correlations between IKBKE mRNA expression of RCC patients with clinical staging, including T, M, N, and grade. (L) The Kaplan–Meier plot of overall survival and progression free-survival for RCC grouped by IKBKE expression. (M) Multivariate Cox regression analysis adjusted for IKBKE expression, age, sex, grade, and stage. (N) The protein expression levels of IKBKE in RCC and normal kidney cell lines by Western blotting. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using the Mann–Whitney U test for (A, B, D, E, and H-K), Student’s t test for (C and G), and log‐rank test for (L). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The corresponding antibodies were employed for IHC: IKBKE (ABclonal, 1:50 dilution) and RRM2 (Proteintech, 1:50 dilution).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, MANN-WHITNEY

Journal: International journal of biological sciences

Article Title: IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway.

doi: 10.7150/ijbs.102666

Figure Lengend Snippet: Figure 2. Oncogenic role of IKBKE in RCC. (A) Evaluation of IKBKE knockdown efficiency in RCC cell lines through Western blotting. (B) The cell proliferation rates in the shControl and shIKBKE groups were assessed in RCC cell lines through a CCK-8 assay. (C-D) The colony numbers in the shControl and shIKBKE groups were compared in RCC cell lines through a colony formation assay. The diameter of each well in panel C is approximately 35 mm. (E) Correlation between IKBKE mRNA expression and Cyclin B1 mRNA expression. (F-G) Cell cycle analysis of RCC cell lines with shControl or shIKBKE through flow cytometry. (H-I) The cell apoptosis of RCC cell lines with shControl or shIKBKE through flow cytometry. (J-L) 786-O cells stably expressing shControl or shIKBKE-specific shRNAs were injected subcutaneously into nude mice for xenograft assays. The xenografts in each group (n = 6 mice per group) were photographed in Panel J, the tumor mass is shown in Panel K, and the tumor growth curve is shown in Panel L. Each bar represents the mean values ± SD of three independent experiments except where stated. Statistical analysis was performed using one-way ANOVA for (B, D, and I), Pearson’s correlation analysis for (E), and Student’s t test for (K and L). ns, no significance, **p < 0.01, ***p < 0.001.

Article Snippet: The corresponding antibodies were employed for IHC: IKBKE (ABclonal, 1:50 dilution) and RRM2 (Proteintech, 1:50 dilution).

Techniques: Knockdown, Western Blot, CCK-8 Assay, Colony Assay, Expressing, Cell Cycle Assay, Flow Cytometry, Stable Transfection, Injection

Journal: International journal of biological sciences

Article Title: IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway.

doi: 10.7150/ijbs.102666

Figure Lengend Snippet: Figure 3. Correlation between IKBKE and sunitinib resistance in RCC. (A) The PI3K–AKT upregulated gene set was used to perform GSEA for the respective data sets with the IKBKE transcription level from TCGA and GSE40435 datasets. (B) The EGFR–Tyrosine-Kinase-Inhibitor-Resistance upregulated gene set was used to perform GSEA for the respective data sets with the IKBKE transcription level from GSE53757 and GSE68417 datasets. (C) IKBKE mRNA expression in sunitinib-escape and paired sunitinib-responsive samples from the GSE76068 dataset. IKBKE mRNA expression in sorafenib-resistant and untreated samples from the GSE64052 dataset. (D) Heatmap analysis showed the top 55 genes involved in sunitinib escape and response in the GSE76068 dataset. (E) PPI networks of IKBKE, RRM2, and 55 genes involved in sunitinib escape and response. (F) RRM2 and IKBKE protein expression levels were measured in 769-P and 786-O cells with shControl or shIKBKE by Western blotting. (G) RRM2 mRNA expression levels were measured in 769-P and 786-O cells with shControl or shIKBKE by qPCR. (H) The correlation between RRM2 expression and IKBKE expression for the data from TCGA database. (I) Representative immunohistochemical staining of RRM2 and IKBKE using the tissue microarray of renal cancer. Scale bars, 100 μm. (J) The correlation between IHC scores of RRM2 and IKBKE (n = 60). Each bar represents the mean values ± SD of three independent experiments except where stated. Statistical analysis was performed using Student’s t test for (C), one-way ANOVA for (G), and Pearson’s correlation analysis for (H and I). ns, no significance, *p < 0.05.

Article Snippet: The corresponding antibodies were employed for IHC: IKBKE (ABclonal, 1:50 dilution) and RRM2 (Proteintech, 1:50 dilution).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Microarray

Journal: International journal of biological sciences

Article Title: IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway.

doi: 10.7150/ijbs.102666

Figure Lengend Snippet: Figure 4. IKBKE binds to RRM2 to activate AKT in RCC. (A) The expression of CyclinB1 and CyclinA was determined by Western blotting in RCC cell lines treated with shIKBKE. (B) The expression of RRM2 was determined by Western blotting in RCC cell lines treated with shIKBKE or MG132. (C) Indicated plasmids were transfected into HEK293T cells and an anti-Flag antibody was used for immunoprecipitation. The precipitates were immunoblotted using Myc- and anti-Flag antibodies. (D) The whole-cell lysates of 769-P and 786-O cells were precipitated with IKBKE antibodies, and the precipitates were examined by immunoblotting to evaluate the endogenous interaction between IKBKE and RRM2. (E) RRM2-Flags were co-transfected with IKBKE plasmids into HEK293T cells, and the whole-cell lysates were subjected to immuno-affinity purification using anti-Flag magnetic beads. The expression of phospho-serine/threonine/tyrosine (p/S/T/Y) was evaluated by Western blotting. (F) 786-O cells were infected with the indicated lentivirus for 72 h, and subsequently, the cells were harvested for Western blotting to determine the expression of target proteins. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using Student’s t test for (E and F). ns, no significance, *p < 0.05, **p < 0.01.

Article Snippet: The corresponding antibodies were employed for IHC: IKBKE (ABclonal, 1:50 dilution) and RRM2 (Proteintech, 1:50 dilution).

Techniques: Expressing, Western Blot, Transfection, Immunoprecipitation, Affinity Purification, Magnetic Beads, Infection

Journal: International journal of biological sciences

Article Title: IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway.

doi: 10.7150/ijbs.102666

Figure Lengend Snippet: Figure 5. IKBKE promotes RCC progression by upregulating RRM2. (A) The expression of IKBKE and RRM2 was measured in RCC cell lines treated with shIKBKE or shRRM2 by Western blotting. (B) The cell proliferation rate of RCC cell lines infected with the indicated lentivirus was evaluated through CCK-8 assays. (C) The expression of IKBKE and RRM2 was measured in RCC cell lines treated with IKBKE or shRRM2 by Western blotting. (D) The cell proliferation rate of RCC cell lines infected with the indicated lentivirus was evaluated through CCK-8 assays. (E-H) The colony numbers of RCC cell lines infected with the indicated lentivirus were evaluated through colony formation assays. The diameter of each well in panel C is approximately 35 mm. (I-J) The cell apoptosis of RCC cell lines infected with the indicated lentivirus was evaluated by Annexin V-7AAD flow cytometry. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA for (B, D, F, and H). ns, no, significance, **p < 0.01, ***p < 0.001.

Article Snippet: The corresponding antibodies were employed for IHC: IKBKE (ABclonal, 1:50 dilution) and RRM2 (Proteintech, 1:50 dilution).

Techniques: Expressing, Western Blot, Infection, CCK-8 Assay, Flow Cytometry

Journal: International journal of biological sciences

Article Title: IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway.

doi: 10.7150/ijbs.102666

Figure Lengend Snippet: Figure 6. The IKBKE–RRM2 axis regulates sunitinib sensitivity in RCC. (A-H) 786-O cells were infected with the indicated lentivirus for 72 h, and subsequently, the cells were treated with or without sunitinib (2 µM). The cell proliferation rate was evaluated through CCK-8 (A, C) and EDU (E and G, scale bars, 50 μm) assays, and cell apoptosis was evaluated by Annexin V-7AAD flow cytometry (B, D). (I-J) 769-P and 786-O cells were infected with the indicated lentivirus for 72 h, and subsequently, the cells were treated with a series of concentrations of sunitinib for 48 h. Cell viability was determined by a CCK-8 assay. (K-M) 786-O cells were infected with the indicated lentivirus for 72 h. After puromycin (5 μg/mL) selection, cells were collected and subcutaneously injected into the nude mice (n = 6 mice per group). These mice were treated with or without sunitinib (20 mg/kg). The xenografts in each group are shown in Panel K, the tumor mass is shown in Panel L, and the tumor growth curve is shown in Panel M. Each bar represents the mean values ± SD of three independent experiments except where stated. Statistical analysis was performed using one-way ANOVA for (A, C, F, H, L, and M). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The corresponding antibodies were employed for IHC: IKBKE (ABclonal, 1:50 dilution) and RRM2 (Proteintech, 1:50 dilution).

Techniques: Infection, CCK-8 Assay, Flow Cytometry, Selection, Injection

Journal: International journal of biological sciences

Article Title: IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway.

doi: 10.7150/ijbs.102666

Figure Lengend Snippet: Figure 7. The IKBKE inhibitor CYT387 regulates sunitinib sensitivity in RCC. (A) 769-P and 786-O cells were treated with or without 5 μM CYT387 for 48 h. The protein expression of IKBKE and RRM2 was detected by Western blotting. (B) 769-P and 786-O cells were treated with control, DMSO or CYT387 (5 μM) for 48 h; subsequently, these cells were harvested and treated with a series of doses of sunitinib for 48 h. Cell viability was determined by CCK-8 assays. (C-F) 786-O cells were treated with DMSO or CYT387 for 48 h. Subsequently, these cells were treated with sunitinib (2 µM). Cell apoptosis was evaluated by Annexin V-7AAD flow cytometry (C, D). The cell proliferation rate was evaluated by EDU assays (E, F). Scale bar = 50 μm. (G-H) 786-O cells were treated with indicated constructs. After 48 h, the cell medium was collected for in vitro angiogenesis assays. Scale bar = 100 μm. Each bar represents the mean values ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA for (D, F, and H). **p < 0.01, ***p < 0.001.

Article Snippet: The corresponding antibodies were employed for IHC: IKBKE (ABclonal, 1:50 dilution) and RRM2 (Proteintech, 1:50 dilution).

Techniques: Expressing, Western Blot, Control, CCK-8 Assay, Flow Cytometry, Construct, In Vitro

Journal: International journal of biological sciences

Article Title: IKBKE regulates renal cell carcinoma progression and sunitinib resistance through the RRM2-AKT pathway.

doi: 10.7150/ijbs.102666

Figure Lengend Snippet: Figure 8. A working model of the IKBKE–RRM2 axis regulating RCC progression and sunitinib resistance. (Left) IKBKE overexpression induces RCC progression and sunitinib resistance by upregulating RRM2 through the RRM2-mediated PI3K/AKT pathway. (Right) IKBKE silencing downregulates RRM2 and induces cell cycle arrest at G2/M phase through the RRM2-mediated PI3K/AKT pathway, suppressing the progression of renal cancer cells and enhancing sunitinib sensitivity. In addition, the IKBKE inhibitor CYT387 can inhibit IKBKE expression and restore sunitinib sensitivity in RCC cells.

Article Snippet: The corresponding antibodies were employed for IHC: IKBKE (ABclonal, 1:50 dilution) and RRM2 (Proteintech, 1:50 dilution).

Techniques: Over Expression, Expressing

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ihc ikbke (ABclonal Biotechnology)

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