American Society of Hematology

Figure 2

$Figure 2. Cdc42 regulates TNFα transcript and protein. (A-B) PMNs were left untreated or transduced with wild-type WASP-CRIB or mutant non–Cdc42-binding WASP-CRIB (10 μg/mL, 25°C, 2 hours) using BioPORTER (BP). Cells were then left untreated or exposed to LPS (2 hours). Supernatants were analyzed by ELISA for TNFα protein (A) and lysates by RT-PCR for TNFα and GAPDH transcripts (B) (*P < .05 compared with buffer/LPS). The results shown are representative of 4 experiments. (C-D) PMNs were left untreated or transduced with GST or L61Cdc42 (10 μg/mL, 25°C, 2 hours) using BP. Cells were then left untreated or exposed to LPS (2 hours). Supernatants were tested by ELISA for TNFα protein (C) and lysates by RT-PCR for TNFα and GAPDH (D). (*P < .001 compared with untreated and GST treated). The gel shown is representative of 3 experiments. (E) PMNs were untreated or transduced with GST or L61Cdc42 (4 hours) and then exposed to buffer or LPS (25 minutes). Cell sonicates were assayed by ELISA for p65 activation (*P < .05) in the presence or absence of competitor (wt) and noncompetitor (mutant) oligonucleotides, and (F) cytoplasmic fractions were immunoblotted for IκBα. A representative immunoblot is shown. PCR gels are representative of 3 independent experiments. (A, C, E) Error bars represent SE.$

Cdc42 regulates TNFα transcript and protein. (A-B) PMNs were left untreated or transduced with wild-type WASP-CRIB or mutant non–Cdc42-binding WASP-CRIB (10 μg/mL, 25°C, 2 hours) using BioPORTER (BP). Cells were then left untreated or exposed to LPS (2 hours). Supernatants were analyzed by ELISA for TNFα protein (A) and lysates by RT-PCR for TNFα and GAPDH transcripts (B) (*P < .05 compared with buffer/LPS). The results shown are representative of 4 experiments. (C-D) PMNs were left untreated or transduced with GST or L61Cdc42 (10 μg/mL, 25°C, 2 hours) using BP. Cells were then left untreated or exposed to LPS (2 hours). Supernatants were tested by ELISA for TNFα protein (C) and lysates by RT-PCR for TNFα and GAPDH (D). (*P < .001 compared with untreated and GST treated). The gel shown is representative of 3 experiments. (E) PMNs were untreated or transduced with GST or L61Cdc42 (4 hours) and then exposed to buffer or LPS (25 minutes). Cell sonicates were assayed by ELISA for p65 activation (*P < .05) in the presence or absence of competitor (wt) and noncompetitor (mutant) oligonucleotides, and (F) cytoplasmic fractions were immunoblotted for IκBα. A representative immunoblot is shown. PCR gels are representative of 3 independent experiments. (A, C, E) Error bars represent SE.

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