Fig. 6
Oxidative stress, hypoxia and estrogen deprivation induces activation of TGFβ and TNFα signaling pathway. Western blots for (a). phospho-SMAD-3 of murine stromal cells treated with H2O2 and CCCP, and (b). phospho-SMAD-2 and (c). TNFα of mouse stromal cells treated with ICI 182780. Nearly confluent mouse stromal monolayers maintained in 10 cm plastic dishes were treated with 100 μM of H2O2 and CCCP for one hour, washed with PBS and incubated in DMEM/10%FCS for 24, 48, or 72 h. Mouse stromal monolayers were also treated with ICI 182780 at different concentrations (10− 8, 10− 7 and 10− 6 M) for 48 and 72 h. Cells were lysed in modified RIPA buffer and analyzed by SDS-PAGE with antibody to mouse phospho-SMAD-2, total SMAD-2, phospho-SMAD-3 and TNFα