Imagequant 5.0 densitometer

Western blotting was performed as described before [6 (link)]. Cells for Western blotting were plated at a density of 2.5 - 3 × 10⁵ cells/mL in 6-well plates. After being cultured in the plates for 24 h, they were subjected to the treatment with LPS, IFN-γ and isoflurane as described above. Cells were lysed and homogenized in 25 mM Tris–HCl, pH 7.4, containing 1 mM EDTA, 1 mM EGTA, 0.1% (vol/vol) α-mercaptoethanol, 1 μM phenylmethylsulfonyl fluoride, 2 μM leupeptin, and 1 μM pepstatin A. The homogenates were centrifuged at 14,000 X g for 10 min at 4°C. The supernatant was used for protein assay with a bicinchoninic acid protein assay kit (Pierce). The protein was loaded at 20 μg protein per lane on a sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then transferred to a polyvinylidene difluoride membrane (Millipore). After incubation with an anti-phospho-p38 antibody(1:1000; Cell Signaling Technology) and an anti-p38 antibody (1:500; Cell Signaling Technology), the protein bands were visualized by using Western blotting detection reagents (GE Healthcare, Piscataway, NJ). The protein bands were densitometrically analyzed by an ImageQuant 5.0 densitometer (Amersham Biosciences, Piscataway, NJ). The results of cells treated with various conditions were then normalized by the data of control cells.

Western blot analysis was performed as described before [8 (link)]. Cells for Western blot were plated at a density of 2.5 – 3 × 10⁵ cells/mL in 6-well plates and plated cells were left to adhere overnight (24 h). After drug treatment with LPS, IFN-γ and morphine, cells were lysed and homogenized in 25 mM Tris–HCl, pH 7.4, containing 1 mM EDTA, 1 mM EGTA, 0.1% (vol/vol) α-mercaptoethanol, 1 μM phenylmethylsulfonyl fluoride, 2 μM leupeptin, and 1 μM pepstatin A. The homogenates were centrifuged at 14,000 × g for 10 min at 4°C. The supernatant was used for protein assay with a bicinchoninic acid protein assay kit (Pierce). The samples were loaded at 20 μg proteins per lane, run on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and then transferred to a polyvinylidene difluoride membrane (Millipore). After incubation with anti-phospho-p38 (1:1000; Cell Signaling Technology), and anti-p38 (1:500; Cell Signaling Technology), the probed protein bands were visualized by using Western blotting detection reagents (GE Healthcare, Piscataway, NJ). The protein bands were densitometrically analyzed by an ImageQuant 5.0 densitometer (Amersham Biosciences, Piscataway, NJ, USA). The results of cells treated with various conditions were then normalized by the data of control cells.