Hs 024

The neutralizing antibodies (nAbs) for MCRs were rabbit anti-human MC1R polyclonal Ab (pAb) (1.25 μg/ml, ab97321; Abcam), rabbit anti-human MC2R pAb (2.5 μg/ml; Millipore), rabbit anti-human MC3R pAb (5 μg/ml, ab21231; Abcam), rabbit anti-human MC4R pAb (5 μg/ml, ab75506; Abcam), goat anti-human MC5R pAb (5 μg/ml, ab92287; Abcam), rabbit anti humanα-MSH pAb (30 μg/ml), and mouse anti-human ACTH monoclonal Ab (mAb) (15 μg/ml, B427; Santa Cruz Biotechnology). Normal rabbit IgG (ab37415; Abcam), goat IgG (ab37373; Abcam), and mouse IgG (Millipore) were used as negative controls. HS 024 (Tocris Bioscience) was used as an antagonist of MC4R, and agouti (1–40)-amide human (Phoenix Pharmaceuticals) was used as an antagonist of MC3R and MC4R.

RO27-3225 (Sigma-Aldrich, St. Louis, MO) as a selective agonist of MC4R was dissolved in saline and tested at three different doses (60 μg/kg, 180 μg/kg, and 540 μg/kg), which were administered intraperitoneally at 1 h after ICH [17 (link), 18 (link)]. HS024 (130 μg/kg; Tocris, Minneapolis, MN) as a specific antagonist of MC4R was dissolved in saline and then administered intraperitoneally 20 min after ICH [26 (link)]. The mice of ICH + vehicle group and ICH + RO27-3225 + saline group received an equal volume of saline. The selective AMPK inhibitor, dorsomorphin (5 μg/mouse; Sigma-Aldrich, St. Louis, MO), was dissolved in 5% dimethyl sulfoxide (DMSO) and then injected intracerebroventricularly 30 min before ICH induction [27 (link), 28 (link)]. The control group was given the same volume of 5% DMSO by intracerebroventricular injection.

Cell culture reagents were obtained from Invitrogen (Darmstadt, Germany). The anti-pERK-1/2 (E-4) and the anti-Rap-1a (sc-1482) antiserum was from Santa Cruz (Heidelberg, Germany). The pCREB-Ser-133 antibody was purchased from cell signaling (Leiden, the Netherlands). The anti-tubulin (clone 6-11B-1) and the peroxidase-conjugated anti-mouse or anti-rabbit antibody, both raised in goat, from Bio-Rad (München, Germany). The firefly luciferase substrate was from Promega (Mannheim, Germany). rp-Br-cAMPs and γ-MSH were from SigmaAldrich (Deisenhofen, Germany). ESI-09, ESI-05 and HJC-0197 were from Biolog (Bremen, Germany), A-812511 from abcam (Cambridge, UK) and PD-184,352, HS-024 or NPY from Tocris (Bristol, UK). α-MSH was from Biotrend (Cologne, Germany) and KT-5720 from Enzo life science (Lörrach, Germany).