(A) Control Lineage 2 (L2C). An aggregate-negative Round 1 (R1) clone (gray box) derived from a starter culture (ST) of cells containing Sup35 NM alone is identified and restreaked to yield progeny Round 2 (R2) clones (gray bracket). 0 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-negative R2 clone (blue box) is restreaked to yield progeny R3 clones (blue bracket). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. An aggregate-negative R3 clone (green box) is restreaked to yield progeny R4 clones (green bracket). 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. The α-His6X and α-Sup35 antibodies recognize the Sup35 NM-mCherry-His6X fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (B) Control Lineage 3 (L3C). As in L2C (A), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones. (C) Control Lineage 4 (L4C). As in L2C (A) and L3C (B), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones.