Sc 7383

U87MG cells were treated with DMSO (control), EB148 or EB54 for different times. When indicated, after 8 h of incubation, cells were washed twice with fresh saline and cultured in drug-free medium for an additional 24 h. At the end of the treatment period, the cells were collected, and suspended in lysis buffer containing 1% of the Protease inhibitor Cocktail. Levels of p53/MDM2 or ERK/MDM2 complexes were quantified using the ELISA test method described above in wells pre-coated with an anti-MDM2 antibody. Cell lysates (20 μg in a final volume of 100 μl) were transferred to the pre-coated wells for 60 min; each well was then incubated for 1.5 h at room temperature with p53 or ERK (sc-7383 SantaCruz Biotechnology, 1:500) primary antibodies.

Gastric corpus mucosa was labeled by immunohistochemistry for proliferation (Ki67 primary antibody, AbCam, Cambridge, UK), apoptosis (cleaved caspase 3, AF835, R and D Systems, Minneapolis, MN, USA), and tyrosine 204 phosphorylation state specific ERK (sc-7383, SantaCruz Biotechnology, Dallas, TX, USA) by immunohistochemistry. All primary antibodies were raised in rabbit and were visualized using the Impress HRP system (Vector laboratories, Peterborough, UK).

The following antibodies were used: mouse monoclonal antibodies to β gal
(1:1,000, G8021, Sigma), pERK (1:50, sc-7383, Santa Cruz), CC1 (1:50, OP80,
Millipore), neurofilament (1:1,000, NE1017, Millipore), and rabbit polyclonal
antibodies to Olig2 (1:500, AB9610, Millipore), ki67 (1:500, SP6, Cell Marque),
Caspr (1:1,000) (ref. 68 (link)), pERK (1:1,000, 4370P,
Cell Signaling), ERK (1:2,000, M5670, Sigma), GPR17 (1:20, Cayman Chemical,
17087), and goat polyclonal antibody to GPR37 (1:50, Santa Cruz, sc-27548), and
rat monoclonal antibodies to MBP (1:300, MAB386, Chemicon), PDGFRα
(1:1,000, APA5, BD Pharmingen), and hybridoma supernatants of mouse anti-O4
(1:5) and rat anti-PLP (1:10, AA3). Secondary antibodies were obtained from
Jackson Immunoresearch and Invitrogen. PLX4032 (ref. 41 (link)) and myr-EPE (ref. 40 (link)) are
generous gifts from Prof. Rony Seger (Weizmann Institute of Science, Rehovot,
Israel). SQ 22536, IBMX and ESI-09 were purchased from Sigma. cAMP levels were
determined by ELISA according to the manufacturer's protocol (cAMP
ELISA kit, Enzo Life Sciences).

We used a rabbit polyclonal antibody directed against a region near the C-terminal of ApoER2 (A3481, Sigma). The rabbit polyclonal antiserum against the recombinant human ApoER2 cytoplasmic domain and the mouse monoclonal anti-HA have been described before [101 (link)]. We also used a p75^NTR rabbit polyclonal antibody (07–476, Millipore), a mouse monoclonal anti-β-tubulin antibody (05–661, Millipore), a mouse monoclonal anti-actin antibody (MAB1501R, Chemicon), a rabbit polyclonal anti-AKT antibody (#9272, Cell Signaling), a rabbit monoclonal anti-phosphorylated AKT antibody (#4060, Cell Signaling), a mouse monoclonal anti-phosphorylated ERK antibody (sc-7383, Santa Cruz Biotechnology), a rabbit polyclonal anti-Dab1 antibody (AB5840, Chemicon International), and a mouse monoclonal anti-MAP2 antibody (MAB378, Chemicon). We used horseradish peroxidase (HRP)-conjugated secondary antibodies (Chemicon) and Alexa 555- and 488-conjugated goat anti-mouse and anti-rabbit secondary antibodies (Molecular Probes).

The murine hepatocyte cell line, AML-12, was purchased through the American Tissue Culture Collection (ATCC) and grown in DMEM:F12 Medium supplemented with 10% FBS, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium (Invitrogen), and 40 ng/mL dexamethasone (MilliporeSigma) (complete medium). For experiments, cells were acclimated to complete medium without dexamethasone for 18 hours prior to stimulation with bacterial LPS (Thermo Fisher Scientific). AML-12 cells were treated with 50 μM MIF098 (45 (link)), ERK activation inhibitor U0126 (Cell Signaling Technologies), or vehicle control (0.1% DMSO; VWR Chemicals LLC) 1 hour prior to LPS challenge at the indicated concentrations. RNA was isolated with the Direct-zol RNA Kit (Zymo Research), and protein was isolated as previously described (14 (link)). Cell lysates were separated on 10% polyacrylamide gels and used for Western blot analysis with antibodies against phospho-ERK1/2 (sc-7383, Santa Cruz Biotechnology Inc.), phospho-p65 (3033S, Cell Signaling Technology), total ERK (06-182, MilliporeSigma), total p65 (6956S, Cell Signaling Technology), and IκBα (9242, Cell Signaling Technology). GAPDH (MAB374, MilliporeSigma) was used as a loading control. Signal intensities were quantified using ImageJ (NIH).

For recombinant proteins, 200 ng protein were mixed with SDS loading buffer and boiled at 100°C for 10 min. For mammalian cells, the protein lysates were prepared by removing the medium, washing with PBS, and adding SDS buffer directly to the plate. Cell lysates were collected and boiled for 10 min. The samples were separated on 4–12% Bis-Tris gels (ThermoFisher Scientific) and transferred to a nitrocellulose membrane. Membranes were probed with anti-ERK (SC154, Santa Cruz Biotechnology) or anti-p-ERK (SC7383, Santa Cruz Biotechnology).