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Other experiments have revealed that only a fraction of hCTR1 is internalized in response to copper, and that hCTR1 internalization is accompanied by its recycling to the plasma membrane when the cells undergo a shift from high to low copper levels [17].
Under these conditions, the Ctr4-VC-Ctr5-VN complexes that were present at the cell surface were internalized in response to copper treatment (Fig. 5A).
Rm1 and Rm2 were not internalized in response to α-MSH during either a 2-h (Fig. 7B and C) or 4-h exposure (not shown).
This combination allowed us to verify that wild-type β2ARs internalized in response to agonist while at the same time binding-defective β2ARs largely remained on the cell surface using two independent methods.
In contrast, the Ctr4 Ctr5 molecules that had been internalized in response to excess copper undergo recycling back to the plasma membrane in response to a transition from high to copper-limiting conditions.
Interestingly, activated rhodopsin of the outer segment is not internalized in response to light [48], suggesting that in PCs, receptors might be sequestered from classical downregulation pathways involving clathrin-mediated endocytosis.
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In the current study and consistent with previous reports [24,58], through ELISA-based internalization assays it was confirmed that hIP internalizes in response to its agonist cicaprost in a time- and concentration-dependent manner.
Rm1 and Rm2 did not internalize in response to α-MSH, in keeping with the inability of α-MSH to activate these RASSLs (Fig. 6B and C).
Most previous studies have concluded that GPCRs internalize in response to agonist as intact dimers, even when only one protomer binds ligand [7], [8], [10], [11].
Although Rm1 did not internalize in response to 1μM THIQ, we observed an approximately 20% decrease in the cell-surface expression of Rm2 in response to the same concentration of THIQ.
Taken together these results are consistent with a model wherein active wild-type β2AR internalizes in response to agonist, while inactive β2AR D113S remains in the plasma membrane (Figure 4A).
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