rabbit anti humanc iap1 antibody  (Cell Signaling Technology Inc)

Journal: Oncotarget

Article Title: Long non-coding RNA CRNDE promotes gallbladder carcinoma carcinogenesis and as a scaffold of DMBT1 and C-IAP1 complexes to activating PI3K-AKT pathway

doi: 10.18632/oncotarget.12023

Figure Lengend Snippet: ( A – B ) Heat map for the Relative expression of si-DMBT1 and si-NC in GBC-SD cells analyzed by using GSEA datasets and bioinformatics predictions. ( C ) RIP analyses were performed using antibodies against DMBT1, with IgG as a negative control in GBC-SD cells. The enrichment of the CRNDE was detected using RT-PCR and normalized to the input. ( D ) RNA pull-down was performed using a CRNDE template and RNA-binding protein separated by SDS-PAGE in GBC-SD cells. The protein bands were excised and detected by mass spectrometry analysis. ( E – F ) C-IAP1 was detected by the Western blotting assay in the samples pulled down by CRNDE. RIP analyses were performed using antibodies against c-IAP1, with IgG as a negative control in GBC-SD cells. The enrichment of the CRNDE was detected using RT-PCR and normalized to the input. ( G – H ) Co-IP assay detected by western blot. IgG as a negative control in GBC-SD cells.

Article Snippet: Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with antibodies against Rabbit anti-human DMBT1 antibody and rabbit anti-humanC-IAP1 antibody (Cell Signaling Technology, CA, USA); antibodies of PI3K-AKT pathway which include: MMP-9, JUK, P-JUK, ERK, P-ERK, AKT, P-AKT(Cell Signaling Technology, CA, USA); normal mouse or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FLAG, mouse anti-G FP (Sigma, CA, USA).

Techniques: Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction, RNA Binding Assay, SDS Page, Mass Spectrometry, Western Blot, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: Long non-coding RNA CRNDE promotes gallbladder carcinoma carcinogenesis and as a scaffold of DMBT1 and C-IAP1 complexes to activating PI3K-AKT pathway

doi: 10.18632/oncotarget.12023

Figure Lengend Snippet: ( A ) Deletion mapping of the DMBT1 domain in binding with CRNDE. RIP assays show association of deletion-DMBT1 domain with CRNDE in GBC-SD cells. Relative enrichment (means ± s.e.m.) represents RNA levels associated with domains relative to an input control from three independent experiments. Antibodies against FLAG and control IgG served as controls. ( B ) GBS-SD cells with stable overexpression of DMBT1 or knockdown of DMBT1 were treated with Actinomycin D (2.5 μM) for 24 h. Detect the mRNA level by RT-PCR. ( C ) Migration numbers of transwell assay of different deletion mapping of the DMBT1 domains. ( D ) Deletion mapping of the C-IAP1-binding domain in CRNDE. Top, diagrams of full-length CRNDE and the deletion fragments. Middle, the in vitro –transcribed full-length CRNDE and deletion fragments show the correct sizes. Bottom, immunoblot analysis for C-IAP1 in protein samples pulled down by the different CRNDE constructs. ( E ) Migration numbers of transwell assay of different deletion mapping of the CRNDE fragments. ( F ) GBS-SD cells with stable overexpression of CRNDE or knockdown of CRNDE were treated with MG132 (5 μM) for 24 h. Cell lysates were immunoprecipitated with antibody against C-IAP1. The precipitates and input were analyzed by immunoblotting.

Article Snippet: Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with antibodies against Rabbit anti-human DMBT1 antibody and rabbit anti-humanC-IAP1 antibody (Cell Signaling Technology, CA, USA); antibodies of PI3K-AKT pathway which include: MMP-9, JUK, P-JUK, ERK, P-ERK, AKT, P-AKT(Cell Signaling Technology, CA, USA); normal mouse or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FLAG, mouse anti-G FP (Sigma, CA, USA).

Techniques: Binding Assay, Control, Over Expression, Knockdown, Reverse Transcription Polymerase Chain Reaction, Migration, Transwell Assay, In Vitro, Western Blot, Construct, Immunoprecipitation

Journal: Oncotarget

Article Title: Long non-coding RNA CRNDE promotes gallbladder carcinoma carcinogenesis and as a scaffold of DMBT1 and C-IAP1 complexes to activating PI3K-AKT pathway

doi: 10.18632/oncotarget.12023

Figure Lengend Snippet: ( A ) RT-qPCR and western blot were used to detect the mRNA levels of CRNDE and protein level of c-IAP1 in knockdown and overexpression DMBT1 cells. ( B ) RT-qPCR was used to detect the mRNA levels of CRNDE and c-IAP1 in knockdown and overexpression DMBT1 cells. ( C ) RT-qPCR was used to detect the mRNA levels of CRNDE and c-IAP1 in knockdown and overexpression DMBT1 cells when knockdown of CRNDE. ( D ) Validation of the RNA-seq results in GBC-SD cells using qRT-PCR. A panel of 4 genes were indeed up regulated by knockdown of DMBT1. ( E ) Four candidate genes (MMP-9, P-JUK, P-ERK, and P-AKT) were selected for western blot analysis. ( F ) luciferase activity of NF-κB pathway in DMBT1-transfected cells was increased, and decreased when overexpression of DMBT1.

Article Snippet: Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with antibodies against Rabbit anti-human DMBT1 antibody and rabbit anti-humanC-IAP1 antibody (Cell Signaling Technology, CA, USA); antibodies of PI3K-AKT pathway which include: MMP-9, JUK, P-JUK, ERK, P-ERK, AKT, P-AKT(Cell Signaling Technology, CA, USA); normal mouse or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FLAG, mouse anti-G FP (Sigma, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Over Expression, Biomarker Discovery, RNA Sequencing, Luciferase, Activity Assay, Transfection

Journal: Oncotarget

Article Title: Long non-coding RNA CRNDE promotes gallbladder carcinoma carcinogenesis and as a scaffold of DMBT1 and C-IAP1 complexes to activating PI3K-AKT pathway

doi: 10.18632/oncotarget.12023

Figure Lengend Snippet: ( A – B ) RT-qPCR was used to detect the mRNA levels of CRNDE in 96 paired GBC tissues. CRNDE significantly upregulated in tumor tissues (β-actin used as an internal control). ( C – D ) RT-qPCR was used to detect the mRNA levels of c-IAP1 in 96 paired GBC tissues. c-IAP1 significantly upregulated in tumor tissues (β-actin used as an internal control). ( E ) Western blot and northern blot assay to detected the DMBT1, C-IAP1 and CRNDE expression level in five paired GBC tissues and NT tissues. ( F ) Correlation between DMBT1 and CRNDE in 42 paired GBC tissues. ( G ) Correlation between CRNDE and c-IAP1 in 42 paired GBC tissues.

Article Snippet: Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with antibodies against Rabbit anti-human DMBT1 antibody and rabbit anti-humanC-IAP1 antibody (Cell Signaling Technology, CA, USA); antibodies of PI3K-AKT pathway which include: MMP-9, JUK, P-JUK, ERK, P-ERK, AKT, P-AKT(Cell Signaling Technology, CA, USA); normal mouse or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FLAG, mouse anti-G FP (Sigma, CA, USA).

Techniques: Quantitative RT-PCR, Control, Western Blot, Northern Blot, Expressing