Figure 1.
263K transformation on changes in replication environment. A) RNA dependency of 263K amplification. 104-fold diluted 263K brain material subjected to amplification in PMCAb conducted in NBH or RNA-depleted NBH. B) Experimental design for testing adaptation of 263K to RNA-depleted NBH and then readaptation to NBH. C) Adaptation of 263K to RNA-depleted NBH. 102-fold diluted 263K brain material was subjected to 9 sPMCAb rounds conducted at 10-fold dilution between rounds and then to 3 rounds at 5-fold dilution between rounds. End products of round 12 for 3 independent sPMCAb reactions are shown and referred to as 263KR−. sPMCAb reactions were conducted in RNA-depleted NBH. Undigested 10% NBH and serial dilution of 263K brain material are shown as references. D) Readaptation of 263KR− to NBH. 263KR− was subjected to 6 sPMCAb rounds with 10-fold dilution between rounds and then to an additional 8 rounds (5 of which are shown) with 100-fold dilution between rounds. sPMCAb reactions were conducted using NBH. 263KR− readapted to NBH is referred to as 263KR+. E) Conformational stability profiles for brain-derived 263K (blue circle), 263KR− (red square), 263KR+ (brown triangle), and brain-derived Drowsy (DY; green diamond). Means ± sd for 3 independent denaturation experiments are shown. F, G) Analysis of PK resistance. F) PK-resistance profiles for brain-derived 263K (blue circle), 263KR− (red square), 263KR+ (brown triangle), and brain-derived DY (green diamond). The data were normalized relative to the intensity of PK-resistant products at 0.05 mg/ml PK. Means ± sd for 3 independent PK-digestion experiments are shown. G) Representative Western blots of 263K, 263KR−, and 263KR+ treated with increasing concentrations of PK as indicated. For conformational stability and PK resistance assays, products of the 12th PMCAb round in RNA-depleted BH were used to assay 263KR−, and products of the 14th PMCAb round in NBH were used to assay 263KR+. All Western blots were stained with 3F4 antibody.