mouse anti‐psrc y416  (Millipore)

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