Cells were rinsed in PBS and scraped in 400 ml of cell lysis buffer containing protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany). Cell lysates were then centrifuged at 12,000 rpm for 15 min at 4°C. The protein concentration in each lysate was then determined using the BCA assay. SDS-PAGE gels were loaded with the same amount of protein, and the NC membrane was blocked for 30 min with skimmed milk. Several primary antibodies were used for western blotting (as 1:1000 dilutions): NF-κB p65, ab16502; IkBα, ab76429; TLR2, ab108998; MyD88, ab219413; IRAK1, ab238; TRAF6, ab33915; TIRAP, ab17218; TAK1, ab109526; p-NF-κB p65, ab76302; IRAK4, 4363; p-PI3K, ab182651 (Abcam, Cambridge, MA, United States). We also used β-actin, 4970, GAPDH, 5174; AKT, 9272; and p-AKT, 4060S (Cell Signaling Technology, United States). HRP-labeled goat anti-rabbit secondary antibodies were used at a dilution of 1:2000 (A0208; Beyotime Biotechnology, Shanghai, China) and immunoreactive bands were detected using an Amersham Imager 600 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
Ab17218
Manufactured by Abcam
Sourced in China, France
Ab17218 is a primary antibody product manufactured by Abcam. It is a research-use only tool that can be used to detect the target antigen in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Gapdh 5174, by Cell Signaling Technology (1 mentions)
Pakt 4060, by Cell Signaling Technology (1 mentions)
β actin 4970, by Cell Signaling Technology (1 mentions)
Akt 9272, by Cell Signaling Technology (1 mentions)
Ab33915, by Abcam (1 mentions)
Ab108998, by Abcam (1 mentions)
Ab76429, by Abcam (1 mentions)
Ab238, by Abcam (1 mentions)
Ab219413, by Abcam (1 mentions)
Ab109526, by Abcam (1 mentions)
Ab76302, by Abcam (1 mentions)
Ab182651, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
A0208, by Beyotime (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Ab77581, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
5 protocols using ab17218
1
Western Blot Analysis of Inflammatory Signaling
2
Immunoblotting Analysis of TLR Signaling Pathway
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The following primary antibodies were used: mouse monoclonal anti-β-Actin (12262, 1:1000, Cell Signaling), mouse monoclonal anti-GAPDH (51332, 1:1000, Cell Signaling), rabbit polyclonal anti-BIG1 (A300-998A, 1:1000, Bethyl), rabbit monoclonal anti-IκBα (4814, 1:1000, Cell Signaling), rabbit monoclonal anti-pS536-p65 (3033, 1:1000, Cell Signaling), rabbit monoclonal anti-pS176/180-IKKα/β (2697, 1:1000, Cell Signaling), rabbit monoclonal anti-TLR4 (14358, 1:1000, Cell Signaling), rabbit monoclonal anti-MyD88 (4283, 1:1000, Cell Signaling), rabbit polyclonal anti-TIRAP (ab17218, 1:1000, Abcam), mouse monoclonal anti-PIP2 (sc53412, 1:200, Santa Cruz), rabbit polyclonal anti-ARF1 (PA1-127, 1:2000, Thermo Fisher), mouse monoclonal anti-ARF3 (610784, 1:500, BD Biosciences), mouse monoclonal anti-ARF5 (H00000381-M01, 1:500, Abnova), rabbit polyclonal anti-ARF6 (ab77581, 1:1000, Abcam), and mouse monoclonal anti-Myc-Tag (2276, 1:1000, Cell Signaling). Chemical reagents used are as follows: LPS (L4516, Sigma-Aldrich; L23351, Invitrogen), R848 (tlrl-r848-5, Invitrogen), CpG ODN (tlrl-2395-5, Invitrogen), FSL-1 (tlrl-fsl, Invitrogen), Pam3csk4 (tlrl-pms, Invitrogen).
3
Validating Prognostic Genes in ccRCC
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According to the expression of prognostic genes in the gene signature in the TCGA and ICGC databases, we selected five hub genes (PYCARD, AIM2, IL6, GSDMB, and TIRAP) that were differentially expressed between the cancer and normal tissues for validation using quantitative real-time PCR (RT-qPCR). Informed consent about the tissue sample analysis was obtained from each patient before the initiation of the study, and the study protocol was approved by the Institutional Review Board of the Second Military Medical University (SMMU) Cancer Center. A total of 40 paired normal and cancer tissues were used to validate the different expression level of model-related genes. For detailed experiment procedures, please refer to the previous literature published by our laboratory. The primer sequences used are listed in Table S2 Supplementary Data https://www.oncomine.org/resource/login.html ) was utilized to validate the different expression of model-related genes in ccRCC and normal tissue.
4
Western Blot Analysis of Lung Protein Expression
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WB analysis of total protein from the lung tissues was performed as previously described (22 (link)). Equal amounts of proteins (50 μg) were electrophoresed in 10% sodium dodecyl SDS-PAGE and transferred onto polyvinylidene difluoride (PDVF) membranes. After blocking in a nonfat milk solution, the PDVF membranes were incubated with primary antibodies specific for TRAF6 (E-AB-18251, Elabscience, Wuhan, China), TIRAP (ab17218, Abcam) and GAPDH (60,004–1-lg, Proteintech, Rosemont, IL) overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (ab97051 or ab6728, Abcam) at room temperature for 1 h. Next, the PVDF membranes were incubated with enhanced chemiluminescence reagent (Merck Millipore, Billerica, MA) before detection using a ChemiScope series 4300 (CLINX Science Instruments, Shanghai, China).
5
Immunofluorescence Staining of pAECs
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pAECs grown on collagen type I-coated coverslips in six-well plates were washed with PBS 1X before fixation with 4% paraformaldehyde (Electron Microscopy Sciences, 15710; can be stored at −80 °C). After permeabilization in PBS 1X/FBS 10%/Triton 0.1% for more than 90 min, the primary antibody against RAGE (1/1000, ab37647, Abcam, Paris, France), HMGB1 (1/400, ab79823, Abcam, Paris, France), Myd88 (1/250, ab133739, Abcam, Paris, France), TIRAP (1/100, ab17218, Abcam, Paris, France), Diaphanous-1 (1/400, ab11173, Abcam, Paris, France), and p65 NF-κB (1/400, 8242, Cell signaling, Saint-Cyr-L’Ecole, France) was applied overnight at 4 °C. After three washes in the permeabilization buffer, the secondary antibody, anti-rabbit Alexa Fluor® 488 (1/1000, A21206, Life Technologies, Villebon-Sur-Yvette, France), was incubated for 2 h at room temperature. Slides were washed three times in PBS 1X and incubated with Hoescht (15 min, dilution in PBS 1X 1/10,000; bisBenzimide H, 33258, Sigma-Aldrich, Saint-Quentin-Fallavier, France). Finally, slides were mounted with CitiFluor™ Tris-MWL 4-88 (Electron Microscopy Science, Hatfield, PA, USA) and examined under a Zeiss LSM800 Airyscan for cells. For the negative controls, incubation without the primary antibody was performed.
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