phosphorylated-glucocorticoid receptor p-gr 4161s  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Anxiolytic effects of Enterococcus faecalis 2001 on a mouse model of colitis

doi: 10.1038/s41598-024-62309-3

Figure Lengend Snippet: Effects of EF-2001 on serum LPS and corticosterone, and p-GR in the brain. Effects of chronic treatment with Enterococcus faecalis 2001 (EF-2001) on increased serum LPS ( a ) and corticosterone ( b ) concentration, and enhanced p-GR in the prefrontal cortex (PFC) ( c ) and ventral hippocampus ( d ) of DSS-treated mice. Quantification of the normalized values of p-GR with t-GR. Original immunoblot images were shown in Supplementary Fig. . ( e ) Microscopy images of p-GR (Ser211) (green), DAPI (blue), and NeuN, GFAP or Iba1 (red) immunostaining in the PFC of DSS-treated mice. Bars represent means ± standard error of mean (SEM). *p < 0.05 and **p < 0.01 vs. water-treated water group, # p < 0.05 and ## p < 0.01 vs. water-treated DSS group (n = 6–8 per group).

Article Snippet: Subsequently, membranes were cut between 50 and 75 kDa, and probed with antibodies except for p-CAMKII, t-CAMKII, and BDNF against phosphorylated (p)-GR (Ser211) (1:1000; Cell Signaling Technology, Danvers, MA, USA, #4161), total (t)-GR (1:200; Santa Cruz Biotechnology, Dallas, TX, USA, sc-393232), NR1 (1:200; Cell Signaling Technology, #4204), NR2A (1:200; Cell Signaling Technology, #4205), NR2B (1:500; Cell Signaling Technology, #4207), p-CAMKII (1:1000; Cell Signaling Technology, #3361), t-CAMKII (1:1000; Cell Signaling Technology, #3362), p-CREB (1:500; Cell Signaling Technology, #9198), t-CREB (1:1000; Cell Signaling Technology, #9197), BDNF (1:1000; Abcam Ltd., Cambridge, UK, ab108319), synaptophysin (1:2000; Sigma-Aldrich, St. Louis, USA, S5768), synaptosomal-associated protein 25 (SNAP25; 1:1000; Santa Cruz Biotechnology, sc-7539), postsynaptic density protein 95 (PSD95; 1:1000; Cell Signaling Technology, #3450), drebrin (1:200; Medical & Biological Laboratories Co., Ltd., Tokyo, Japan, D029-3), microtubule-associated protein 2 (MAP2; 1:2000; Sigma-Aldrich, M9942), neuronal nuclei (NeuN; 1:500; Millipore, MAB377), and β-actin (1:1000; Santa Cruz Biotechnology, sc-47778) overnight at 4 °C.

Techniques: Concentration Assay, Western Blot, Microscopy, Immunostaining

Journal: Molecular psychiatry

Article Title: Corticosteroid Sensitization Drives Opioid Addiction

doi: 10.1038/s41380-022-01501-1

Figure Lengend Snippet: (a) Male and female rats were prepared with intravenous (IV) catheters and trained to self-administer (SA) methadone (unit dose: 300 μg/kg/0.1 ml) in 1 h ShA sessions (FR1) and split into ShA and LgA (12 h) groups. We used the unit dose of 300 μg/kg/0.1 ml based on studies that reported that rats dose-dependently self-administered methadone and that methadone substitutes for morphine [21–23]. We used the 300 μg/kg/0.1 ml dose for both sexes based on their similar response to methadone-induced antinociception and hyperalgesia during withdrawal [24]. Once rats in the LgA group significantly escalated their methadone self-administration, rats in both the ShA and LgA groups were acutely administered with mifepristone (0, 30, 60, and 90 mg/kg; intraperitoneal, 90 min before testing) in a within-subjects Latin-square design. (b) Methadone infusions (± SEM) in rats in the ShA and LgA groups. ***p < 0.0001, difference from session 1. ShA: n = 18 (10 males, 8 females). LgA: n = 18 (9 males, 9 females). To confirm that the rats in the LgA group exhibited greater signs of opioid dependence than rats in the ShA group, we tested the rats in the von Frey (VF) test to measure hyperalgesia during spontaneous withdrawal and naloxone-precipitated signs of somatic withdrawal (STW). (c) Phosphorylation of GRs at Ser232 in the CeA in rats in the LgA group relative to rats in the ShA group. The optical densities of total GR bands were normalized to the housekeeping protein GAPDH. Because there was no significant difference in total GR levels between the ShA and LgA groups, we normalized the optical densities of phosphorylated GR bands to the optical densities of total GR bands. ***p < 0.001, difference from ShA. ShA: n = 14 (7 males, 7 females). LgA: n = 12 (6 males, 6 females). (d, e) Methadone infusions (± SEM) in (d) rats in the ShA group and (e) rats in the LgA group. **p < 0.01, ***p = 0.0002, difference from 0 mg/kg (Dunnett’s post hoc comparisons). ShA: n = 18. LgA: n = 18.

Article Snippet: The primary antibodies were phosphorylated GR (Ser 211 ) antibody (1:500, catalog no. 4161, Cell Signaling, Danvers, MA, USA), GR monoclonal antibody (1:500, catalog no. MA1–510, Invitrogen, Waltham, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2500, catalog no. AB9485, Abcam, Cambridge, MA, USA).

Techniques: Phospho-proteomics