Ab131438

RPMI-1640 medium, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and ox-LDL were purchased from Invitrogen (Shanghai, China). miR-16 mimic, miR-16 inhibitor and siRNA targeting PDCD4 (PDCD4 siRNA) were purchased from GenePharma (Shanghai, China), as well as their negative control (NC) oligonucleotide duplex that did not target any gene. Antibodies against p38 (ab7952), phosphorylated (p-)p38 (ab4822), extracellular signal-regulated kinase (ERK; ab180163), p-ERK (ab131438), c-Jun NH2-terminal kinase (JNK; ab199380), p-JNK (ab18680), p65 (ab90532) and PDCD4 (ab79405) were obtained from Abcam (Cambridge, MA, USA).

After protein extraction from brain tissues, proteases and phosphatases were inhibited by the addition of a Complete Mini Protease Inhibitor Cocktail Tablet (Roche, Basel, Switzerland) and the phosphatase inhibitor phenylmethylsulfonyl fluoride (10 mM). The protein concentrations of the samples were assessed using the Bradford protein assay. Total proteins (50 µg) were heated at 90°C for 10 minutes and then separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred onto nitrocellulose membranes, which were blocked using 5% skim milk. The membranes were then incubated with primary polyclonal antibodies recognizing EPOR (1:1000, FNab02816, Wuhan Fine Biotech, Wuhan, China), p-ERK1 (1:500, ab131438, Abcam, Cambridge, United Kingdom), ERK (1:1000, ab115799, Abcam), and CD34 (1:1000, AF-4117, R&D Systems, Minneapolis, MN) at 4°C overnight. Secondary antibodies (1:5000, ab6721 [Abcam] or 1:5000, sc-2352 [Santa Cruz, Dallas, TX]) were then reacted with the bound primary antibodies and visualized using enhanced chemiluminescence reagents (Pierce, Rockford, IL). The loading control comprised glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Image-J software was used for densitometry analysis.