2016
DOI: 10.1038/srep27316
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Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model

Abstract: G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-ba… Show more

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Cited by 12 publications
(11 citation statements)
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“…After addressing the key challenges of the eSiMBlot assay, we tested the power of this approach to directly visualize multisite PTM codes in individual protein molecules. For these proof-of-principle experiments, we used in vitro autophosphorylated recombinant EGFR (rEGFR) tagged with enhanced green fluorescence protein (eGFP), SNAP-tag, 20 and FLAG antigen sequences, 21 isolated by affinity purification with anti-FLAG beads ( Figure 4 a). EGFR is a distinct subfamily of receptor tyrosine kinases.…”
Section: Resultsmentioning
confidence: 99%
“…After addressing the key challenges of the eSiMBlot assay, we tested the power of this approach to directly visualize multisite PTM codes in individual protein molecules. For these proof-of-principle experiments, we used in vitro autophosphorylated recombinant EGFR (rEGFR) tagged with enhanced green fluorescence protein (eGFP), SNAP-tag, 20 and FLAG antigen sequences, 21 isolated by affinity purification with anti-FLAG beads ( Figure 4 a). EGFR is a distinct subfamily of receptor tyrosine kinases.…”
Section: Resultsmentioning
confidence: 99%
“…The HEK cell line stably expressing 3HA‐hCB 1 receptors has been described previously (Cawston et al, ). This cell line was used as the background to generate a HEK cell line that co‐expresses CB 1 and D 2 receptors, and is first described in Hunter et al (). The HEK cell line stably expressing the human 3HA‐hCB 2 receptor has also been described previously (Grimsey et al, ).…”
Section: Methodsmentioning
confidence: 99%
“…A panel of six chemically distinct CB 1 agonists was compiled to allow delineation of ligand-dependence of the two signalling pathways. line was used as the background to generate a HEK cell line that co-expresses CB 1 and D 2 receptors, and is first described in Hunter et al (2016). The HEK cell line stably expressing the human 3HA-hCB 2 receptor has also been described previously (Grimsey et al, 2011).…”
Section: Introductionmentioning
confidence: 99%
“…A secondary anti-mouse antibody conjugated to HRP (Santa Cruz Biotechnologies, Dallas, TX, USA) was used to detect the presence of the anti-myc tag antibody. The FLAG tag was present in each construction but we were unable to detect this epitope perhaps due to the post-translational sulfatation of this epitope leading to poor recognition by the specific antibody 22 .…”
Section: Methodsmentioning
confidence: 86%