Figure 7.

Involvement of FHC in the inhibitory effects of opioids on CXCR4 signaling. A, Protein level of FHC are reduced in neurons transfected with FHC siRNAs as determined by immunoblot. Control neurons are transfected with a scrambled siRNA. B, Effect of 1 μm DAMGO on CXCL12-induced ERK/Akt phosphorylation was determined in control or FHC siRNA transfected neurons using phosphospecific antibodies and Western blot. Membranes were stripped and reprobed for total proteins to confirm equal loading. C, Protein levels of FHC are reduced in brain areas expressing FHC shRNA as reported in detail in Materials and Methods. Top blots: GenScript shRNA plasmid; bottom blots: Open Biosystem shRNA plasmid. L, Left cortex (i.e., transfected side); R, right cortex. Quantification of the band densities revealed 76% reduction in FHC tissue levels in the left hemispheres (see also supplemental Fig. 2D, available at www.jneurosci.org as supplemental material). Membranes were stripped and reprobed for actin to confirm equal loading. D, Representative pictures of FHC immunostaining in cortical brain sections of electroporated pups (6 d after transfection, GenScript plasmid). E, Pups (P0–P1) were treated with saline or morphine acutely (single injection, 20 mg/kg, s.c.) and killed within 24 h. Brain slices were treated with CXCL12 (50 nm) and processed for GTPγS autoradiography. Analysis was performed in the cortex (medial and lateral cortex), distinguishing between left and right side in the case of electroporated animals. In controls, no differences have been noticed between left and right areas of the brain (data not shown). Data are expressed as mean ± SEM of five animals per experimental group (^#p < 0.01 vs saline; *p < 0.01 vs left cortex).