Ab 108

For Il6 neutralizing experiments Il6 neutralizing antibody (R&D, AB-406-NA) and control IgG antibody (R&D, AB-108) were used to assess effects of secreted Il6 on the proliferation of MEFs. The Stat3 inhibitor WP1066 (Selleckchem, S2796), PI3K inhibitor LY294002 (Cell Signaling, #9901), Rapamycin (Cell Signaling, #9904) and JAK inhibitor I (Calbiochem, 420099) were resuspended in DMSO. JQ1 was a kind gift from Dr. J. Bradner (Harvard Medical School) and was used as described previously (31 (link)). Note that the IC₅₀ value for inhibition of proliferation was 1.4 μM for Pten/Trp53-deleted cells, and 3.3 μM for wt cells. These results are in similar range to earlier reported, WP1066 effects on cell survival, proliferation and Stat3 phosphorylation (54 (link)–56 (link)).

Conditioned medium was depleted of CXCL1 or CCL2 using antibodies previously loaded onto protein A/G Plus-Agarose beads (Santa Cruz Biotechnology, Dallas TX) at a ratio of 10 μL beads to 1μg antibody. Beads were washed before and after antibody loading. Antibodies were purified anti-CXCL1 (R and D systems MAB453, Minneapolis MN) used at 2 μg/mL conditioned medium, anti-CCL2 (R and D systems AF-479) at 5 μg/mL, Rat IgG2a isotype control (R and D systems MAB006) at 2 μg/mL, or goat IgG (R and D systems AB-108) at 5 μg/mL. Incubation with conditioned medium was on a rocking platform overnight at 4°C, after which the beads were pelleted and the depleted conditioned medium was collected and used for downstream applications.