Anti phospho erk1 2 antibody

Raw 264.7 macrophage cells (ATCC) were cultured in RPMI 1640 containing 10% FBS and 100 U/ml penicillin/streptomycin. PZ-HPV-7 cell (ATCC) were cultured in Keratinocyte Serum Free media containing 0.05 mg/mL bovine pituitary extract and 5 ng/mL human recombinant EGF. Cells were maintained in a 37 °C incubator supplemented with 5% CO₂. To obtain Raw 264.7-conditioned media, 2.5 × 10⁵ cells per 6-well dish were grown in Keratinocyte Serum Free complete media, and supernatant was collected. Conditioned media were freshly prepared for each experiment. Both anti-F4/80 and anti-cyclin D1 antibodies were from Thermo Fisher Scientific; anti-iNOS and anti-CD206 antibodies were obtained from Abcam. The anti-CD38 antibody was from Novus Biologicals; anti-Akt, anti-phospho-Akt (ser473) and anti-ERK antibodies were purchased from Cell Signal Technology. The anti-phospho-ERK1/2 antibody was from Santa Cruz Biotechnology, and the anti-Ym1 antibody was obtained from STEMCELL Technologies. Human recombinant CCL3, CCL4, CXCL2, IL-1ra, M-CSF1 and GDNF were purchased from PeproTech. Human recombinant osteopontin was obtained from BioLegend. Matrigel was from Corning. U0126 and wortmannin (WT) were obtained from Cell Signaling Technology. Other reagents used are described in the specific experiment sections.

After indicated treatment, Spred2 over-expressed or slicenced K562 cells were collected and the protein was extracted. Then, the expression of Spred2 was detected by rabbit anti-human Spred2 antibody (Sigma). And, the activation of MAPK signalling pathway was detected by anti-phospho-ERK1/2 antibody and anti-ERK-1/2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 6h, 12h, 18h, 24h after treatment with 10mg/ml PMA or at 1h after treated by 0.1, 0.5 or 1.0μM imatinib.