Importantly, H89, a moderately specific inhibitor for PKA [31, 32], was employed in the present study to investigate the role of CREB or p-CREB
, activated by PKA.
The effect of 100-Hz EA may be associated with BDNF activation at the VTA, while a cumulative effect of multiple 100-Hz stimulation may be related to acceleration of dynorphin synthesis and down-regulation of p-CREB
However, the ERK1/2 inhibitor completely suppressed the activation of p-ERK1/2, p-CREB
, and Bcl-2 and promoted the activation of Bax and cleaved caspase-3 in TK-treated group, indicating that ERK1/2 may be the main upstream protein in this signaling pathway activated by TK.
In addition, each of the monotherapies negatively regulated several proteins that are central to ATP-consuming processes, such as processes that involve p-CREB
and TORC2, resulting in the downregulation or inhibition of gluconeogenesis.
The mechanisms linking opioid receptor activation to p-CREB
are not clear.
Many studies have indicated that [alpha]-MSH is a c-AMP activator, stimulating the protein levels of p-CREB
and MITF to enhance the synthesis of tyrosinase-related proteins leading to melanogenesis .
The role of P-CREB
in food intake, especially as related to opioid exposure, is less well studied.
p-CREB/CREB Untreated 0.37 [+ or -] 0.02 0.36 [+ or -] 0.04 0.97 [+ or -] 0.07 * 1.5 Gy 0.32 [+ or -] 0.02 0.43 [+ or -] 0.05 1.30 [+ or -] 0.09 6 Gy 0.31 [+ or -] 0.01 0.48 [+ or -] 0.05 1.50 [+ or -] 0.09 * [FIGURE 5 OMITTED]
Immunofluorescence was used to evaluate the levels of cAMP, phosphorylation of PKA (p-PKA), and p-CREB
expression in the right ACC.
Western blotting was performed using skin tissues for detecting (a) p-CREB
, CREB, MITF, and TFIIB in nucleus fraction and (d) tyrosinase and [beta]-actin in cytosol fraction (n = 4/group).
To study the immunofluorescent histochemistry of p-CREB
and GFAP, the lumbar enlargement of the spinal cord was dissected out after the perfusion and fixed in the same, fresh fixative overnight at 4[degrees]C.
BDNF (SantaCruz Biotechnology, sc-546, 1 : 200), P-CREB
(Cell Signaling Technology, 9198s, 1:500), CREB (Cell Signaling Technology, 9197, 1: 500), PKA (Proteintech, 55388-1-AP, 1:1000), NMDAR1 (Cell Signaling Technology, 5104s, 1:1000), NR2A (Cell Signaling Technology, #4205, 1 : 1000), NR2B (Cell Signaling Technology, 4212s, 1:1000), and tubulin (Proteintech, 10094-1-AP, 1:2000) were used at 4[degrees]C overnight.