Figure 1.
Figure 1. Relationship between LMP1 and CD95 expression. (A) Kinetics of LMP1 and CD95 expression by Western blot in EBV latency III EBNA2-regulatable EREB2-5 cells: EREB2-5 cells were starved for estrogen for 48 hours to arrest the EBV latency III program. Readdition of estrogen reinduced EBNA2 activity and subsequent expression of the whole set of latency III viral genes. 0h indicates estrogen deprived; 3h, 8h, 24h, and cont indicate 3, 8, and 24 hours, and continuous estrogen exposition, respectively. Ponceau red staining of an 18-kDa band is shown (PR). After background subtraction, the CD95 signal was normalized to Ponceau red signal. Fold changes in CD95 protein were then calculated using the Optiquant system and are presented in the histogram. Fold change values are indicated at the top of each bar when compared with the estrogen-deprived condition. (B) Flow cytometry detection of LMP1CT induction in LCL cells transfected with pRT-1-LMP1CT after treatment of the cells with 1 μg/mL doxycycline for 36 hours. The percentage of EGFP-positive cells is indicated in the upper-right corner of the histogram. (C) CD95 mRNA expression by real-time RT-PCR (fold changes are indicated at the top of each bar when compared with the untreated condition) in EREB2-5 cells stably transfected with the pRT1-LMP1wt vector, in the absence (-) or presence (+) of doxycycline or estrogen (upper panel) and in LCL cells stably transfected either with pRT1-LMP1CT coding for a dominant-negative form of LMP1 or with the pRT1-LMP1wt–inducible vector (lower panel). Error bars correspond to the standard deviation of 3 experiments. (D) LMP1 and CD95 protein expression by Western blot in LCL and estrogen-deprived EREB2-5 cells stably transfected either with the pRT1-LMP1CT vector or with the pRT1-LMP1wt vector, in the absence (-) or presence (+) of doxycycline. As in Figure 1A, the CD95 signal was normalized to Ponceau red (PR) signal, and fold changes in CD95 protein are presented in the histogram. (E) Expression of surface membrane CD95 by flow cytometry in LCL cells transfected with either pRT1-LMP1CT or pRT1-LMP1wt vector. Percentage of positive cells for CD95 in the doxycycline-treated condition is indicated in the upper-right corner of the histogram.

Relationship between LMP1 and CD95 expression. (A) Kinetics of LMP1 and CD95 expression by Western blot in EBV latency III EBNA2-regulatable EREB2-5 cells: EREB2-5 cells were starved for estrogen for 48 hours to arrest the EBV latency III program. Readdition of estrogen reinduced EBNA2 activity and subsequent expression of the whole set of latency III viral genes. 0h indicates estrogen deprived; 3h, 8h, 24h, and cont indicate 3, 8, and 24 hours, and continuous estrogen exposition, respectively. Ponceau red staining of an 18-kDa band is shown (PR). After background subtraction, the CD95 signal was normalized to Ponceau red signal. Fold changes in CD95 protein were then calculated using the Optiquant system and are presented in the histogram. Fold change values are indicated at the top of each bar when compared with the estrogen-deprived condition. (B) Flow cytometry detection of LMP1CT induction in LCL cells transfected with pRT-1-LMP1CT after treatment of the cells with 1 μg/mL doxycycline for 36 hours. The percentage of EGFP-positive cells is indicated in the upper-right corner of the histogram. (C) CD95 mRNA expression by real-time RT-PCR (fold changes are indicated at the top of each bar when compared with the untreated condition) in EREB2-5 cells stably transfected with the pRT1-LMP1wt vector, in the absence (-) or presence (+) of doxycycline or estrogen (upper panel) and in LCL cells stably transfected either with pRT1-LMP1CT coding for a dominant-negative form of LMP1 or with the pRT1-LMP1wt–inducible vector (lower panel). Error bars correspond to the standard deviation of 3 experiments. (D) LMP1 and CD95 protein expression by Western blot in LCL and estrogen-deprived EREB2-5 cells stably transfected either with the pRT1-LMP1CT vector or with the pRT1-LMP1wt vector, in the absence (-) or presence (+) of doxycycline. As in Figure 1A, the CD95 signal was normalized to Ponceau red (PR) signal, and fold changes in CD95 protein are presented in the histogram. (E) Expression of surface membrane CD95 by flow cytometry in LCL cells transfected with either pRT1-LMP1CT or pRT1-LMP1wt vector. Percentage of positive cells for CD95 in the doxycycline-treated condition is indicated in the upper-right corner of the histogram.

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