Monoclonal mouse antibodies to gapdh and α tubulin

THP-1 monocytes (ATCC TIB-202) and primary human monocytes/macrophages were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% heat-inactivated Fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO₂. THP-PPM1A and THP-ΔPPM1A cells were generated previously^{13 (link),14} . Human PBMCs were isolated from buffy coats by the Ficoll-Paque density centrifugation method. Monocytes were enriched by positive selection using anti-CD14 mAb-coated microbeads from Miltenyi Biotec (San Diego, CA) according to manufacturer’s protocol. Primary monocytes were differentiated with either 5 ng/ml GM-CSF (R&D Systems, Minneapolis, MN) or 10 ng/ml M-CSF (StemCell Technologies, Vancouver, Canada) for 5–11 days to obtain monocyte derived macrophages (MDM). Fetal bovine serum was obtained from Life Technologies (Grand Island, NY). Phorbol ester 13-phorbol-12-myristate acetate (PMA), puromycin, bryostatin, imiquimod, and Pam3CSK4 were purchased from Sigma (St. Louis, MO). Monoclonal PPM1A antibody were purchased from Thermo Scientific (Rockford, IL). Monoclonal mouse antibodies to GAPDH and α-tubulin were purchased from Santa Cruz (Dallas, Texas) and Cell Signaling (Danvers, MA), respectively.

THP-1 monocytes (ATCC TIB-202) and primary monocytes were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO₂. THP-PPM1A cells were generated previously21 (link). Human PBMCs were isolated from buffy coats by the Ficoll-Paque density centrifugation method. Monocytes were enriched by positive selection using anti-CD14 mAb-coated microbeads from Miltenyi Biotec (San Diego, CA) according to manufacturer’s protocol. Monocytes were then differentiated with 5 ng/ml GM-CSF (R&D Systems, Minneapolis, MN) for 5–7 days to obtain monocyte derived macrophages (MDM). fetal bovine serum was obtained from Life Technologies (Grand Island, NY). Phorbol ester 13-phorbol-12-myristate acetate (PMA), puromycin, etoposide, kanamycin and anisomycin were purchased from Sigma (St. Louis, MO). Sanguinarine and SP600125 were purchased from Fisher Scientific (Pittsburgh, PA). CellTiter-Glo luminescent cell viability assay kit was purchased from Promega (Madison, WI). Polyclonal PPM1A antibodies were purchased from Thermo Scientific (Rockford, IL). Monoclonal mouse antibodies to GAPDH and α-tubulin were purchased from Santa Cruz (Dallas, Texas) and Cell Signaling (Danvers, MA), respectively.

THP-1 monocytes (ATCC TIB-202), Jurkat T cells (ATCC TIB-152) and primary monocytes were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO₂. Human PBMCs were isolated from buffy coats by the Ficoll-Paque density centrifugation method. Monocytes were enriched by positive selection using anti-CD14 mAb-coated microbeads from Miltenyi Biotec (San Diego, CA) according to manufacturer's protocol. fetal bovine serum was obtained from Life Technologies (Grand Island, NY). The phorbol ester 13-phorbol-12-myristate acetate (PMA), puromycin and LPS were purchased from Sigma (St. Louis, MO). Recombinant human Interferon-gamma was purchased from Genscript (Piscataway, NJ). PE-conjugated antibodies to human CD4, CD14, HLA-DR, and ICAM-1 were purchased from BD Biosciences (San Diego, CA). Anti-CD16-PE and anti-PPM1A antibodies were purchased from Thermo Scientific (Rockford, IL). Anti-TLR5-PE antibody was purchased from ABCAM (Cambridge, MA). Monoclonal mouse antibodies to GAPDH and α-tubulin were purchased from Santa Cruz (Dallas, Texas) and Cell Signaling (Danvers, MA), respectively.