MIP-1δ induces expression and activity of c-fos and NFATc1 via stimulation of the PLCγ2 and NF-κB signaling pathways.

<p>BMM were treated with or without MIP-1δ (0.1 pg/ml). (A) c-fos and NFATc1 protein expression were determined by Western analysis performed on equal amounts of protein from total cell lysates. (B) Activation of AP-1 was determined 30 minutes following treatment with MIP-1δ by non-radioactive EMSA analysis of nuclear cell lysates using AP-1 oligonucleotides. Oct-1 served as a loading control and 200-fold excess unlabeled oligonucleotide (200X-U.O.) served as a competitive control. (C) NFATc1 protein expression was determined 60 minutes following treatment with MIP-1δ by Western analysis performed on equal amounts of protein from nuclear cell lysates. (D) CTSK, TRAP, and DC-STAMP mRNA expression were determined by qRT-PCR 24 hours following treatment with MIP-1δ. Data are representative of three independent experiments performed in triplicate, expressed as the mean (n = 9) ± s.e.m, and were analyzed by unpaired, two-tailed Student’s t-test. <i>p</i><0.01; ***, <i>p</i><0.001. (E, F) BMM were treated with MIP-1δ (0.1 pg/ml) alone or in combination with the (E) NF-κB inhibitor IKK2 inhibitor VIII (IKK2-I) or the (F) PLC inhibitor U73122 and protein expression was determined 60 minutes (c-fos, NFATc1) or 30 minutes (IκBα, phospho-PLCγ2) following MIP-1δ treatment by Western analysis performed on equal amounts of protein from total cell lysates. (E) Densitometry values are provided below respective lanes for ease of interpretation. Data are representative of at least two independent experiments.