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Figure S1 Characterization of phosphosite-specific antibodies using phosphatase treatment of receptors. (A and B) HEK293 cells stably expressing the μ-opioid receptor (MOR) were either not exposed or exposed to 10 μM DAMGO (DAMGO). The cells were lysed and proteins were either dephosphorylated (+) using lambda protein phosphatase or not dephosphorylated (−). Samples were then immunoblotted with the anti-pS363 antibody {3199} (pS363) or the anti-pS375 antibody {2493} (pS375) at a concentration of 0.1 μg·mL−1. Blots were stripped and reprobed with the phosphorylation-independent anti-MOR antibody {UMB-3} to confirm equal loading of the gel (MOR). Shown are representative results from one of three independent experiments. The position of molecular mass marker is indicated on the left (in kDa).

Figure S2 Time-course of S375 dephosphorylation in morphine-treated cells. (A and B) HEK293 cells stably expressing the μ-opioid receptor (MOR) were either exposed to 10 μM morphine for 30 min; washed three times with either cold PBS (pH 7.4) (washout) or cold citrate buffer (pH 4.5) (acid washout); and incubated in the absence of agonist for 0, 10, 20, 40 or 60 min. Cells were lysed and immunoblotted with the anti-pS375 antibody {2493} (pS375). Blots were stripped and reprobed with the phosphorylation-independent anti-MOR antibody {UMB-3} to confirm equal loading of the gel (MOR). Note that washout under acidic conditions facilitated S375 dephosphorylation after morphine treatment. Representative results form one of at least three independent experiments are shown. The position of molecular mass marker is indicated on the left (in kDa).

FilenameFormatSizeDescription
BPH_1382_sm_Fig_S1.tif650KSupporting info item
BPH_1382_sm_Fig_S2.tif386KSupporting info item

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