Mab3241

Neutralizing antibodies to human CCL3 (MAB270), CCL14 (MAB3241) and CCL2 (MAB679), and mouse CCL3 (AB-450-NA), CCL2 (AB-479-NA) were purchased from R&D Systems. Immunohistochemistry antibodies to human CCL3, CCL14 and CCL2 and mouse F4/80 were purchased from Santa Cruz Bio. Western blot antibodies against cyclin D1, p27Kip1, c-myc, pAkt, Akt, pErk, Erk, IL6, c-myc, pp38MAPK, p38MAPK, and α-actin were from Cell Signaling. FITC-, PE, and APC-conjugated monoclonal antibodies to human CD14, CD68, CCR1, CCR2, CCR5 and mouse CD14, CD138, F4/80 were from purchased Biolegend.
The CellTrace CFSE kit was purchased from Invitrogen. Cell proliferation was analyzed using an MTS kit (Promega) following the standard protocol. Propidium iodide (PI) and RNase were purchased from Sigma Aldrich.

Protein extracts were probed with antibodies against human UHRF1 (sc-373750, Santa Cruz biotechnology, USA), KLF6 (sc-7158, Santa Cruz biotechnology, USA), COX-2 (A5787, ABclonal, USA), CSF1 (AF216, R&D Systems, USA), DNMT1 (sc-271729, Santa Cruz biotechnology, USA), and CCL14 (MAB3241, R&D Systems, USA) or β-actin (A5441, Sigma, USA). Stained blots were visualized with chemiluminescence assays using ECL detection reagents (Millipore, USA).

For PGE₂ neutralization, TAMs (10⁵) isolated from human HCC patients were cultured in a well of a 6-well plate with 2 mL RPMI medium for 24 hours. The supernatants were collected and mixed with 2 μg/mL PGE₂ neutralizing antibody (anti-PGE₂, 360150, Cayman Chemical, USA) or isotype IgG for 24 hours. HepG2 or Huh7 cells were incubated with the supernatants for 24 hours before analysis. For neutralization of CSF1 and CCL14, the supernatants were harvested from 10⁷ HepG2 cells stably expressing UHRF1 (or transfected with control vector) that were cultured in 10-cm dishes for 48 hours. The supernatants were mixed with 2 μg/mL of CSF1 antibody (AF216, R&D Systems, USA), CCL14 antibody (MAB3241, R&D Systems, USA) or isotype IgG for 24 hours. These supernatants were used to treat TAMs for 24 or 48 hours before analysis.