Journal: PLoS ONE

Article Title: c-Src Regulates Akt Signaling in Response to Ghrelin via β-Arrestin Signaling-Independent and -Dependent Mechanisms

doi: 10.1371/journal.pone.0004686

Figure Lengend Snippet: A, Effect of siRNA depletion of c-Src on ghrelin-induced Akt phosphorylation. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM) at 37°C. After stimulation, equal amounts of protein in each sample were used to assess the expression of c-Src (left panel) and Akt phosphorylation (right panel) by western blotting. Expression of c-Src was quantified by densitometry and expressed as percentages of the level of c-Src in control siRNA-transfected cells (mean±S.E.). Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation at HM (S473) and A-loop (T308) after ghrelin addition to control siRNA-transfected cells (mean±S.E.). B, Effect of ghrelin on Y phosphorylation of Akt and interaction between Akt and c-Src. Cells were incubated with ghrelin (100 nM, 5 min) at 37°C, lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with pAkt HM(S473), pY, pSrc (Y416) antibodies. C, Effect of ghrelin on Y phosphorylation of Akt in the presence of c-Src siRNA. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM, 5 min) at 37°C. Equal amounts of protein in each sample were used to assess the expression of c-Src [upper panel; values shown (mean±S.E.) are percentages of the level of c-Src in control siRNA-transfected cells]. Cells were lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with with pAkt HM(S473), pY, pSrc (Y416) antibodies. Immunoblots are representative of three independent experiments.

Article Snippet: Anti-p44/42 MAPK rabbit polyclonal, anti-pSrc (Y416) rabbit polyclonal, anti-pAkt HM (S473), anti-pAkt A-loop (T308), anti-Akt rabbit polyclonal, anti-Rictor rabbit polyclonal, anti-mTOR rabbit polyclonal and anti-pPDK1 (S241) rabbit polyclonal antibodies were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Transfection, Expressing, Western Blot, Control, Incubation, Immunoprecipitation

Journal: PLoS ONE

Article Title: c-Src Regulates Akt Signaling in Response to Ghrelin via β-Arrestin Signaling-Independent and -Dependent Mechanisms

doi: 10.1371/journal.pone.0004686

Figure Lengend Snippet: A, Effects of the Src inhibitor PP2 and its negative control, PP3, on the ghrelin-induced Akt activation in 3T3-L1 preadipocyte cells. Serum-starved cells were pretreated with PP2 (5 µM, 30 min) or PP3 (5 µM, 30 min) before ghrelin stimulation (100 nM) for the indicated time periods. Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation at HM (S473) and A-loop (T308) (mean±S.E of three independent experiments). B, Effect of ghrelin on the assembly of complexes containing β-arrestins 1 and 2 and pAkt. Preadipocyte 3T3-L1 cells were incubated with ghrelin (100 nM, 10 min) at 37°C, lysed and immunoprecipitated (IP) with antibodies to β-arrestins 1 and 2, and then analyzed by western blotting with pAkt HM (S473), pAkt A-loop (T308), pY, pSrc (Y416) antibodies. For A and B, western blots are representative of three independent experiments.

Article Snippet: Anti-p44/42 MAPK rabbit polyclonal, anti-pSrc (Y416) rabbit polyclonal, anti-pAkt HM (S473), anti-pAkt A-loop (T308), anti-Akt rabbit polyclonal, anti-Rictor rabbit polyclonal, anti-mTOR rabbit polyclonal and anti-pPDK1 (S241) rabbit polyclonal antibodies were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Negative Control, Activation Assay, Phospho-proteomics, Incubation, Immunoprecipitation, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Src‐dependent phosphorylation of μ‐opioid receptor at Tyr 336 modulates opiate withdrawal

doi: 10.15252/emmm.201607324

Figure Lengend Snippet: WT mice were implanted with either placebo or morphine pellets as detailed in the . Low (×20) and high‐magnification (×60) photomicrographs showing the colocalization of pMOR Y336 and pSrc Y416 in the TH + neurons of the LC (arrows) from a placebo‐and‐naloxone‐treated mouse (Placebo + Naloxone), a morphine‐and‐naloxone‐treated mouse (Morphine + Naloxone), and a morphine‐and‐saline‐treated (Morphine + Saline) mouse. The green, red, and magenta colors represent TH, pSrc Y416 , and pMOR Y336 , respectively. Scale bar = 70 μm. The specificity of the pMOR Y336 and pSrc Y416 antibodies was confirmed by the disappearance of pMOR Y336 and pSrc Y416 immunoreactivity when pMOR Y336 and pSrc Y416 antibodies were pre‐incubated with the immunoprecipitated MOR complex that had been extracted from the LC of morphine‐dependent WT mice with naloxone‐precipitated withdrawal (Morphine + Naloxone). Scale bar = 70 μm. The bar graph shows the colocalization analysis of the WT, MOR −/− , and Fyn −/− mice (Fig ). The percentage of colocalized pMOR Y336 , pSrc Y416 , and TH results from the quantification in three mice/genotype, four slices/mouse. Significant differences among the groups (WT, MOR −/− , and Fyn −/− ) were determined using one‐way ANOVA, followed by Duncan's post hoc comparison. *** P = 0.0001 MOR −/− versus WT; *** P = 0.0001 Fyn −/− versus WT; ## P = 0.002729 significant differences between MOR −/− and Fyn −/− . The photomicrographs show minimal colocalization between pMOR Y336 and pSrc Y416 in the TH + neurons of the LC from MOR −/− or Fyn −/− mice treated with chronic morphine (pellets for 3 days) and injected with naloxone after morphine pellets removal on the 4 th day. The green, red, and magenta colors represent TH, pSrc Y416 and pMOR Y336 , respectively. Data are presented as means ± SEM. Scale bar = 70 μm. The phosphorylation of MOR Y336 (pMOR Y336 ) and Src Y416 (pSrc Y416 ) was not detected within the MOR complex in the LC and the hippocampus of WT mice after acute injection of morphine (30 min, 10 mg/kg). An affinity‐purified polyclonal antibody against the MOR N‐terminus (conjugated to Dynabeads) was employed to IP pSrc and pMOR as described in the and Fig A. The immunoblots were probed with anti‐pSrc Y416 (1:1,000), anti‐pMOR Y336 (1:500), or anti‐G β (Santa Cruz sc‐378, 1:500). Source data are available online for this figure.

Article Snippet: The sections were incubated with either rabbit anti‐TH (1:1,000, for the identification of Fyn shRNA injections and WT MOR or MORY336F injections), goat anti‐TH (GeneTex GTX113016, 1:1,000), mouse anti‐pSrc Y416 (Millipore clone 9A6 #05‐677, 1:500), or peptide affinity‐purified rabbit anti‐pMOR Y336 (1:500) overnight at 4°C.

Techniques: Incubation, Immunoprecipitation, Injection, Affinity Purification, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Src‐dependent phosphorylation of μ‐opioid receptor at Tyr 336 modulates opiate withdrawal

doi: 10.15252/emmm.201607324

Figure Lengend Snippet: The midbrain was dissected from mice that had been implanted with placebo or morphine pellets and i.c.v. injected with saline or AZD0530. An affinity‐purified polyclonal antibody against the MOR N‐terminus (conjugated to Dynabeads) was employed to IP pSrc and pMOR as described in the . The immunoblots were probed with anti‐pSrc Y416 (1:1,000), anti‐pMOR Y336 (1:500), or anti‐G β (Santa Cruz sc‐378, 1:500). In the middle panel, a Western blot analysis shows the disappearance of the pMOR Y336 immunoreactivity when the pMOR Y336 antibody was pre‐incubated with the corresponding phospho‐peptide (GeneTex PJ90076 NPVL(pY)AFLDENC). This pre‐adsorption control was performed with the immunoprecipitated MOR complex that had been extracted from the LC of morphine‐dependent WT mice with naloxone‐precipitated withdrawal. On the right, another Western blot shows residual Src activities in the Fyn −/− mice (Src needs to be phosphorylated at Tyr 416 before activation). The reduced bands correlate with the sparsely detectable pSrc Y416 immunoreactivity in the photomicrographs of Figs C and B. This should be a clear indication of the specificity of the anti‐pSrc Y416 . The bar graphs show the densitometric quantification and statistical analysis of the changes in pMOR Y336 from the experiment presented in (A, left panel). The bar graphs show the densitometric quantification and statistical analysis of the changes in pSrc Y416 from the experiment presented in (A, left panel). The bar graphs show no significant differences in the densitometric quantification of G β (from the experiment presented in A, left panel) among the conditions (the internal control, n = 3 mice). Data information: Data are presented as means ± SEM. In (B and C), the density of each band was normalized against G β (the internal control), and the result from the placebo with naloxone group (lane 1) was set to 100%. The experiments were repeated three times. Significant differences between the various treatment groups ( n = 3 mice/group) (lanes 2–5) and the control ( n = 3 mice) (lane 1) were determined using Student's t ‐test. ### P = 0.001 Placebo + Naloxone versus MS + Naloxone in both (B and C), *** P = 0.00086 and *** P = 0.00077 MS + Naloxone versus MS + Naloxone + AZD in (B and C), respectively.

Article Snippet: The sections were incubated with either rabbit anti‐TH (1:1,000, for the identification of Fyn shRNA injections and WT MOR or MORY336F injections), goat anti‐TH (GeneTex GTX113016, 1:1,000), mouse anti‐pSrc Y416 (Millipore clone 9A6 #05‐677, 1:500), or peptide affinity‐purified rabbit anti‐pMOR Y336 (1:500) overnight at 4°C.

Techniques: Injection, Affinity Purification, Western Blot, Incubation, Adsorption, Immunoprecipitation, Activation Assay

Journal: EMBO Molecular Medicine

Article Title: Src‐dependent phosphorylation of μ‐opioid receptor at Tyr 336 modulates opiate withdrawal

doi: 10.15252/emmm.201607324

Figure Lengend Snippet: WT, MOR −/− , or Fyn −/− mice were implanted with either placebo or morphine pellets and administered naloxone as detailed in the A, B Minimal colocalization between pMOR Y336 and pSrc Y416 was observed in the TH + neurons of the LC from MOR −/− (A) or Fyn −/− (B) mice treated with chronic morphine (pellets for 3 days) and injected with naloxone after morphine pellet removal on the 4 th day. The green, red, and magenta colors represent TH, pSrc Y416 , and pMOR Y336 , respectively. C Colocalization of pMOR Y336 (green) and pSrc Y416 (red) in the hippocampus of WT mice implanted with either placebo or morphine pellets. The yellow denotes overlap. D A magnified view of the CA1 (upper) and CA3 (lower) regions from merged images of morphine‐ and naloxone‐treated animals. Data information: Scale bar = 70 μm for the LC and 100 μm for the hippocampus.

Article Snippet: The sections were incubated with either rabbit anti‐TH (1:1,000, for the identification of Fyn shRNA injections and WT MOR or MORY336F injections), goat anti‐TH (GeneTex GTX113016, 1:1,000), mouse anti‐pSrc Y416 (Millipore clone 9A6 #05‐677, 1:500), or peptide affinity‐purified rabbit anti‐pMOR Y336 (1:500) overnight at 4°C.

Techniques: Injection

Journal: EMBO Molecular Medicine

Article Title: Src‐dependent phosphorylation of μ‐opioid receptor at Tyr 336 modulates opiate withdrawal

doi: 10.15252/emmm.201607324

Figure Lengend Snippet: WT mice were either implanted with placebo or morphine pellets for 4 days. Saline or AZD0530 (50 μg/side) was injected into the LC 30 min prior to naloxone administration as detailed in the . Immunostaining of TH (green), pSrc Y416 (red), or MOR Y336 (magenta) in the LC following the injection of saline (upper) or AZD0530 (lower) into the LC. Scale bar = 70 μm. Immunostaining of pSrc Y416 (red) or MOR Y336 (green) in the hippocampus following the injection of saline (upper) or AZD0530 (lower) into the LC. The yellow denotes overlap. Scale bar = 100 μm. Source data are available online for this figure.

Article Snippet: The sections were incubated with either rabbit anti‐TH (1:1,000, for the identification of Fyn shRNA injections and WT MOR or MORY336F injections), goat anti‐TH (GeneTex GTX113016, 1:1,000), mouse anti‐pSrc Y416 (Millipore clone 9A6 #05‐677, 1:500), or peptide affinity‐purified rabbit anti‐pMOR Y336 (1:500) overnight at 4°C.

Techniques: Injection, Immunostaining