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Figure 5 | BMC Immunology

Figure 5

From: Cytokine generation, promoter activation, and oxidant-independent NF-κB activation in a transfectable human neutrophilic cellular model

Figure 5

Transient overexpression of proteins in neutrophil-like PLB-985 cells. (A) DMSO-differentiated PLB-985 (day 5) were nucleofected with a plasmid encoding human PKCα or with vector alone (lane 1). The cells were cultured for 6 or 12 h after nucleofection, and processed for immunoblot analysis of their PKCα content. Membranes were then reblotted with a p38 MAPK antibody (as a loading control). (B) DMSO-differentiated PLB-985 (day 5) were nucleofected with a plasmid encoding a dominant negative form of human IκB-α (S32,36A), or with empty vector ("mock"). The cells were cultured for 6 h after nucleofection, and stimulated for 45 min with 100 ng/ml LPS or 100 U/ml TNFα, prior to immunoblot determination of their IκB-α or actin content. (C) DMSO-differentiated PLB-985 (day 5) were nucleofected with a plasmid encoding human IκB-α (S32,36A), or with empty vector ("mock"). The cells were cultured for 6 h after nucleofection, and stimulated for another 6 h in the absence ("resting") or presence of 100 ng/ml LPS or 100 U/ml TNFα. Culture supernatants were then harvested and analyzed in ELISA for the indicated cytokines. Results shown in this figure are representative of three independent experiments.

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