Journal: Scientific Reports

Article Title: Agonist-induced phosphorylation bar code and differential post-activation signaling of the delta opioid receptor revealed by phosphosite-specific antibodies

doi: 10.1038/s41598-020-65589-7

Figure Lengend Snippet: DPDPE-induced DOP receptor phosphorylation is mediated by GRK2 and GRK3. ( A ) HEK293 cells stably expressing HA-hDOP receptor were stimulated with 1 µM DPDPE, 1 µM PMA or 10 µM forskolin for 10 min at 37 °C. Cell lysates were immunoblotted with anti-pT361 or anti-pS363 antibodies. Blots were stripped and reprobed with the anti-HA antibody as loading control. Blots are representative, n = 4. (B) Cells described in ( A ) were preincubated with either vehicle (DMSO; -) or compound (cmpd) 101 at the indicated concentrations for 30 min at 37 °C, then treated with buffer or 1 µM DPDPE for 10 min at 37 °C. Lysates were immunoblotted as described in ( A ). Blots are representative, n = 3. (C,D) Cells described in ( A ) were transfected with siRNA targeted either to (C) GRK2, GRK3, or GRK2 and GRK3 (GRK2/3), (D) GRK5, GRK6, or GRK5 and GRK6 (GRK5/GRK6), or non-silencing siRNA control (SCR) for 72 h, then stimulated with1 µM DPDPE for 10 min at 37 °C. Lysates were immunoblotted as described in (A). Knock down of GRKs was confirmed by Western blot (bottom panels in C and D). Densitometry readings, above the blots, were normalized to SCR (control) transfected cells, which were set to 100%. Data are means ± SEM from three to five independent experiments. *p < 0.05 vs. SCR control by one-way ANOVA with Bonferroni’s post-hoc test.

Article Snippet: Anti-GRK2 (sc-562), anti-GRK3 (sc-563), anti-GRK5 (sc-518005) and anti-GRK6 (sc-566) antibodies were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Control, Transfection, Knockdown, Western Blot