P erk1 2 monoclonal antibody

TMA construction was previously described in detail [29 (link)]. Immunohistochemistry was performed with PLA2G16 antibody at a 1:100 dilution (Item No. 10337, Cayman Chemical, Ann Arbor, USA) and p-ERK1/2 monoclonal antibody at a 1:300 dilution (#4370, Cell Signaling Technology, Boston, USA).

Aliquots containing 30 μg of protein were blotted as previously described7 (link)8 9 (link). Briefly, protein samples were analyzed on 10% SDS–PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) in a wet transfer unit (Bio-Rad, Hercules, CA, USA). Membranes were with 5% non-fat dry milk at room temperature for 1 h and incubated overnight at 4 °C with the following primary antibodies: GPR91 polyclonal antibody (1:1000), p-ERK1/2 monoclonal antibody (1:3000, Cell Signaling Technology, Boston, MA, USA), ERK1/2 monoclonal antibody (1:3000, Cell Signaling Technology, Boston, MA, USA), c-Fos monoclonal antibody (1:1000), C/EBP β monoclonal antibody (1:1000), Hypoxia inducible factor-1α (HIF-1α) monoclonal antibody (1:200, abcam, Cambridge, MA, USA), VEGF monoclonal antibody (1:1000, abcam, Cambridge, MA, USA), β-actin monoclonal antibody (1:5000, Sigma Aldrich, Saint Louis, MO, USA). β-Tublin monoclonal antibody (1: 1:1000, abcam, Cambridge, MA, USA) and Histone H3 monoclonal antibody (1:1000, ProteinTech Group, Chicago, IL, USA). Membranes were washed with TBS-Tween 20 and incubated with the appropriate HRP-conjugated secondary antibodies (1:1000, ProteinTech Group, Chicago, IL, USA) for 1 h at room temperature. Bands were visualized using an enhanced ECL detection kit (Bio-Rad, Hercules, CA).