PSN cells were metabolically labeled with [9,10-3H]palmitic acid (0.5 mCi/ml) for 1–6 h at 37 °C in N-MEM, containing 1 mM sodium pyruvate to inhibit palmitate metabolism through fatty acid -oxidation 28 ,29 (link). After labeling, cells were washed with SP buffer and lysed in radioimmunoprecipitation assay buffer (RIPA; 10 mM sodium phosphate, 150 mM NaCl, 2 mM EDTA, 50 mM sodium fluoride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.2). Lysates were then immunoblotted with anti-DDK (FLAG) monoclonal antibody (Clone OTI4C5, Origene) to determine NHE1 levels and volumes containing equal amounts of NHE1 (verified by a second immunoblot) were immunoprecipitated with anti-DDK (FLAG) monoclonal antibody (Clone OTI4C5, Origene) bound to protein A beads. Precipitated NHE1 was resolved on 4–20% SDS-polyacrylamide gels, treated with Fluoro-Hance (Research Products International) fluorographic reagent for 30 min, dried, and exposed to x-ray film for ~ 60 days at −80°C.
Anti ddk flag monoclonal antibody
Manufactured by OriGene
The Anti-DDK (FLAG) monoclonal antibody is a laboratory tool used for the detection and purification of proteins that are tagged with the DDK (FLAG) epitope sequence. This antibody specifically binds to the DDK (FLAG) tag, allowing for the identification and isolation of the target protein from complex samples.
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4 protocols using anti ddk flag monoclonal antibody
1
Metabolic Labeling of NHE1 Protein
2
Transcription Factor Binding Assay
3
Immunoprecipitation and Immunoblotting of DDK-tagged Proteins
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Forty-eight hours after transfection, cells were harvested and whole-cell extract was prepared as described above. Extracts were incubated overnight with 1 μg of anti-DDK (FLAG) monoclonal antibody (Origene) (Supplemental Table 7 ). The following day, Protein G Magnetic Beads (Surebeads, Bio-Rad) were added. After 1 hour of incubation, the beads were magnetized using a DynaMag-2 magnet (Invitrogen, Thermo Fisher Scientific) and washed several times with PBS 0.1% Tween-20. Bolt LDS Sample Buffer (4×, Novex) was used as an elution buffer, and finally denaturation was performed at 95°C for 3 minutes. Concurrent with the immunoblotting of the obtained eluate, a tenth of the amount of the whole-cell extract used for the IP was blotted as the input condition. Immunoblotting for FLAG antibody, COPB2, and COPE was performed as described above (Supplemental Table 7 ).
4
Immunohistochemical Analysis of Xenograft Tumors
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Paraffin‐embedded tumor specimens from the xenograft experiments study were immunostained as described previously [29 (link), 36 ]. Details of the antibodies used are provided in Supplementary materials and methods . In brief, paraffin sections (2 μm thick) on poly‐l ‐lysine‐coated slides were incubated with an anti‐DDK (FLAG) monoclonal antibody (dilution 1:1000, OriGene). After blocking endogenous peroxidase activity with 3% v/v of 30% hydrogen peroxide stock solution (Sigma Merck‐Aldrich) and performing microwave antigen retrieval in sodium citrate buffer (pH 6), primary antibodies were detected using the EnVision, HRP‐labeled System (DAKO, from Agilent, Santa Clara, CA, USA) and visualized using 3,3'‐diaminobenzidine as substrate. For negative controls, the primary antibodies were replaced by isotype‐ and concentration‐matched irrelevant antibodies. Conventional histological staining (H&E, Agilent) was performed on paraffin sections (2 μm thick) in order to evaluate the overall vascularization of tumor tissues.
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