American Society of Hematology

Fig. 6.

$Fig. 6. Regulation of Bcl-2, Bcl-X, A1, and Bax in CD34+ cells upon PECAM-1 interactions. / (A) Cells were allowed to adhere to PECAM-1+ transfectants (D12P, lane 3) or to mock-transfected NIH3T3 fibroblasts (mock, lane 2) for 30 minutes, cultured for 36 hours, lysed, and 50 μg of protein per lane was loaded and run on a 12% SDS-PAGE. Lys (lane 1) shows cells before adhesion. After electrotransfer, samples were blotted with the specific anti–Bcl-2, Bcl-X, Bax, and anti–PECAM-1/CD31 mAbs, followed by GAM-HRP, and developed with ECL plus (Amersham). One representative experiment of 4 experiments performed is shown. (B) Normalized aliquots of total RNA were extracted from cells before (CTR, lane 1) or after migration through HUVECs (lane 3), D12P (lane 4), naked filters (lane 5), or upon cross-linking of PECAM-1/CD31 with the specific M89D3 mAb (lane 6), or with control Ig followed by GAM (lane 7). Lane 2: cells treated with 100 ng/mL LPS. Amplification was then performed by PCR with the specific primers for A1 or β-actin and PCR products were size-fractionated by 8% acrylamide electrophoresis. (C) Bands corresponding to A1 or β-actin in panel B were subjected to densitometric analysis and results expressed as pixel number (au).$

Regulation of Bcl-2, Bcl-X, A1, and Bax in CD34⁺ cells upon PECAM-1 interactions.

(A) Cells were allowed to adhere to PECAM-1⁺ transfectants (D12P, lane 3) or to mock-transfected NIH3T3 fibroblasts (mock, lane 2) for 30 minutes, cultured for 36 hours, lysed, and 50 μg of protein per lane was loaded and run on a 12% SDS-PAGE. Lys (lane 1) shows cells before adhesion. After electrotransfer, samples were blotted with the specific anti–Bcl-2, Bcl-X, Bax, and anti–PECAM-1/CD31 mAbs, followed by GAM-HRP, and developed with ECL plus (Amersham). One representative experiment of 4 experiments performed is shown. (B) Normalized aliquots of total RNA were extracted from cells before (CTR, lane 1) or after migration through HUVECs (lane 3), D12P (lane 4), naked filters (lane 5), or upon cross-linking of PECAM-1/CD31 with the specific M89D3 mAb (lane 6), or with control Ig followed by GAM (lane 7). Lane 2: cells treated with 100 ng/mL LPS. Amplification was then performed by PCR with the specific primers for A1 or β-actin and PCR products were size-fractionated by 8% acrylamide electrophoresis. (C) Bands corresponding to A1 or β-actin in panel B were subjected to densitometric analysis and results expressed as pixel number (au).

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