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. 2005 Dec 19;171(6):1013–1022. doi: 10.1083/jcb.200504137

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Copyright © 2005, The Rockefeller University Press

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Figure 6. — Heterozygosity at the lpr locus rescues the apoptotic defects of Raf-1 KO PEFs in culture. (A) Fas expression is reduced in the lpr/+ background, but Fas is organized in clusters at the surface of Raf-1 KO PEFs. Cells in suspension were treated with αFas as described in Fig. 2 A. Fas clustering was detectable in 68 ± 3% of KO cells and in 31 ± 2% of c-raf-1 ^–/– ;lpr/+ cells. (B) Fas expression is reduced in the lpr/+ background. The expression of Raf-1, Fas, and FasL in whole PEF lysates was analyzed by immunoblotting. An ERK immunoblot is shown as a loading control. Molecular mass markers are shown in kilodaltons on the left. (C) FasL mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker in base pairs. (D) c-raf-1 ^–/– ;lpr/+ (KO;lpr/+) PEFs successfully accumulate in culture. WT, WT;lpr/+, KO, and KO;lpr/+ PEFs were cultured in DME/10% FCS. Cell numbers were determined at the indicated times. The values are the mean ± SD (error bars) of four individual batches of PEFs/genotype, each assayed in triplicate. *, P < 0.04; **, P < 0.025; ***, P < 0.01 according to a t test, all compared with KO. (E) Spontaneous apoptosis in continuously growing WT, WT;lpr/+, KO, and KO;lpr/+ PEFs. Asynchronous cells were stained with propidium iodide, and their DNA content was determined by FACS analysis. The percentage of apoptotic cells (DNA content < 2n) is indicated. (F) PEFs were treated with 500 ng/ml αFas plus 1 μg/ml Chx for 22 h. Cell death was assessed as in Fig. 1 A. The values in E and F are the mean ± SD (error bars) of at least three individual batches of PEFs/genotype. (E) *, P < 0.01. (F) *, P < 0.025 according to a t test, all compared with KO. (G) Defective internalization in KO and KO;lpr/+ MEFs. Internalization was determined as described in Fig. 2 B and expressed as the percentage of the internalization occurring in WT cells. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025 according to a t test, all compared with WT.