Figure 5. Western blot analyses of protein extracts from astrocytes. A representative figure from three repeating experiments is shown. A: Analysis is shown of protein extracts from rat brain cortex astrocytes that were subjected to different durations of pressure. Control cells were allowed to remain in the incubator at 37 °C without being exposed to any increase in pressure. Other astrocytes were subjected to an increase in pressure. Cells were subjected to fixed pressure and then analyzed for iso[4]LGE₂–protein modification. About 3,000 isolated rat brain cortex astrocytes were subjected to a pressure of 100 mmHg for different time periods (ranging from 3–33 h). Following pressure treatment, the culture medium was immediately replaced with fresh medium, and the cells were incubated at 37 °C for 16 h at atmospheric pressure. B: Western blot analysis is shown of protein extracts from astrocytes that were subjected to different post pressure recovery periods for iso[4]LGE₂ modification. About 3,000 isolated rat brain cortex astrocytes were subjected to a pressure of 100 mmHg for a period of 3 h and were then allowed to recover at 37 °C. Following pressure treatment, the culture medium was immediately replaced with fresh medium, and the cells were incubated at 37 °C for varying periods (ranging from 1–10 days) at atmospheric pressure. Western blot analysis was performed using rabbit polyclonal antibody to iso[4]LGE₂–protein adduct after fractionation of total cell lysates (25 μg protein lysate was loaded in each lane) on 4%–20% SDS–PAGE and transfer to a PVDF membrane. In A and B, bottom panels were probed with anti-GAPDH as indicated.