Fig. 2. cf50^– cells do not have cf50 mRNA or CF50 protein. (A) A northern blot of RNA from vegetative cells was probed with a fragment of the cf50 cDNA. A 0.9 kB band (arrow) is present in Ax2 wild-type parental cells but not in the cf50^– cells. Aliquots of the samples were electrophoresed on separate gels and stained with ethidium bromide to verify equal loading and that the ribosomal bands were not degraded. (B) A western blot of vegetative cells was stained with anti-CF50 antibodies. A 50 kDa band is present in Ax2, countin^– and smlA^– cells, but is absent from cf50^– cells. (C) A western blot of conditioned starvation media from the above cell lines was stained with anti-CF50 antibodies. A 50 kDa band is present in Ax2, countin^– and smlA^– CMs, but is absent from cf50^– CM. (D) A similar blot stained with anti-countin antibodies, showing countin present in the wild-type and smlA^– CMs, absent from the countin^– CM and present at low levels with what appears to be a lower molecular mass band in the cf50^– CM. (E) A blot of conditioned HL5 growth media showing that CF50 is secreted from growing Ax2, countin^– and smlA^– cells, but not from cf50^– cells. (F) A similar blot of conditioned HL5 growth media stained with anti-countin antibodies. For B-F, SDS-polyacrylamide gels of aliquots of the samples used for the western blots were stained with Coomassie Blue to verify that equal amounts of protein were loaded for each blot.