C5a-induced increase in cytosolic IL-8 mRNA. Freshly prepared PBMC (0.5 × 10⁶) were preincubated with either the transcription inhibitor actinomycin D, the NF-κB inhibitor PDTC, or PTX for 1 hour (lanes 4, 5, and 6), as specified on top of each lane. The concentrations of the inhibitors used in this study were the same as indicated in Fig 3. The cells were then stimulated with 200 nmol/L of human C5a for 2 hours or with 40 ng/mL of TNF (lane 2) as a positive control. Total RNA was extracted from the cell pellets, separated in formaldehyde/agarose gel (10 μg/lane) and subjected to Northern blot analysis using a ³²P-labeled IL-8 cDNA fragment as probe. The autoradiograph shows specific bands of IL-8 transcript. The same blot was stripped and reprobed with³²P-labeled β-actin for equal loading control.