Figure 2.

Lentiviral Overexpression of Human TP in Cultured Reticulocytes

(A and C) CD34⁺ hematopoietic stem cells and expanding BEL-A cells were transduced using lentivirus with TP cDNA generating cTP (A) and bTP (C) cells and subsequently differentiated into reticulocytes. TP expression was assessed at different time points during differentiation by flow cytometry, where at each indicated time point 1 × 10⁵ cells were fixed and permeabilized and subsequently labeled with a TP antibody. Expression is normalized to endogenous expression of TP at the proerythroblast stage (N = 6 ± SEM). (B) Expression by western blotting of TP, Band3, and GAPDH during differentiation. Equal cell numbers were loaded (1 × 10⁶ cells per lane). (D) TP activity was measured in 1 × 10⁶ cTP cells using the spectrophotometry-based assay at the indicated time point during differentiation (N = 6). (E) Time course of TP activity in whole cells using the spectrophotometry-based assay. Decrease in thymidine concentration was measured in the supernatant of 1 × 10⁶ cells at the indicated time points (N = 3).