Determination of TNF-α receptor expression levels in transduced LSK BM by flow. Transduced murine BM cells were isolated by flow sort and were cultured for 7 days. Cells were then stained with antibodies directed against lineage markers, c-Kit, Sca-1, and either TNFR1 or TNFR2. (A) The mean percentage of TNFR1⁺ or TNFR2⁺ LSK cells and (B) the mean fluorescent intensity of staining for TNFR1 or TNFR2 within different retroviral transduced populations. Data represent the least squares mean estimates plus or minus SEM from mixed-effects model analysis of 3 or 4 independent experiments. □ represents Fancc^−/− + SF-IG; , Fancc^−/− + SF-FAC-IG; ■, Fancc^−/− + SF-HOXB4-IG; , WT + SF-IG. *Significant after multiple comparison adjustment (P < .05) by Tukey method. (C,D) 32D cells were transduced with either SF-IV or SF-HOXB4-IV, and Venus⁺ cells were isolated by flow sorting 2 days after transduction and expanded for 4 days in vitro. Expanded cells were harvested and treated either with 20 ng/mL TNF-α (+) or with an equivalent volume of PBS (−). After 5 minutes of incubation at 37°C, cells were harvested and lysed. The lysate from 2 × 10⁵ cells was probed by immunoblot using an antibody specific for (C) NF-κB p65 phosphorylated at the Ser536 residue or (D) SAPK/JNK phosphorylated at the Thr183/Tyr185 residues. Membranes were then sequentially stripped of antibody and reprobed with antibodies against either total NF-κB p65 and then β-actin; or total SAPK/JNK and then β-actin.