Human mcp 1 ccl2 elisa max deluxe set

The CaP cells were collected and lysed with lysis buffer (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm Na₂ EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm beta-glycerophosphate, 1 mm Na₃VO₄, 1 μg/ml leupeptin) with 1 mm phenylmethylsulfonyl fluoride (PMSF). The cell lysates were centrifuged and supernatant was used as proteins. After separating of 25–50 μg protein in SDS-PAGE, samples were incubated with CCL2 (Thermo Fisher Scientific, Waltham, MA, USA), β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA), p-ERK and ERK (Cell Signaling Technology, MA, USA). For CCL2, p-ERK, ERK immunoblots, 1:1000 diluted antibody solutions in 5% skim milk was used. For the β-actin immunoblot, 1:10 000 diluted antibody solutions were used. All antibody solutions were incubated overnight at 4 °C. Following the incubation with appropriate secondary antibody, immunoblot was analysed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA).
For ELISA, LNCaP cells were treated with recombinant WNT5A and culture medium was collected and centrifuged after 48 h. The level of CCL2 was measured with Human MCP-1/CCL2 ELISA MAX Deluxe Set (Biolegend, San Diego, CA, USA).