Figure 1. Expression and targeted inactivation of Fbw7 isoforms. (A) Fbw7 isoform mRNA abundance in the indicated cell types. (B) 293A cells were cotransfected with vectors encoding FLAG-tagged Fbw7 (0.3 μg Fbw7α and 3 μg each of Fbw7β and Fbw7γ) and treated with cycloheximide (CHX) as indicated. The three isoforms are indicated. (C) Schematic demonstrating locations of inserted stop codons for each knockout genotype (see text). (D) Lysates were prepared from confluent p150 plates of Hct116 clones of the indicated genotypes and immunoprecipitated with polyclonal anti-Fbw7 antibody followed by immunoblotting with monoclonal anti-Fbw7 antibody. (E) Real-time PCR analysis of relative Fbw7 isoform mRNA expression in Hct116 Fbw7 knockout cells. Results are expressed as relative to wild-type Hct116 cells. Error bars show standard deviation (three replicates).
Figure 1.

Expression and targeted inactivation of Fbw7 isoforms. (A) Fbw7 isoform mRNA abundance in the indicated cell types. (B) 293A cells were cotransfected with vectors encoding FLAG-tagged Fbw7 (0.3 μg Fbw7α and 3 μg each of Fbw7β and Fbw7γ) and treated with cycloheximide (CHX) as indicated. The three isoforms are indicated. (C) Schematic demonstrating locations of inserted stop codons for each knockout genotype (see text). (D) Lysates were prepared from confluent p150 plates of Hct116 clones of the indicated genotypes and immunoprecipitated with polyclonal anti-Fbw7 antibody followed by immunoblotting with monoclonal anti-Fbw7 antibody. (E) Real-time PCR analysis of relative Fbw7 isoform mRNA expression in Hct116 Fbw7 knockout cells. Results are expressed as relative to wild-type Hct116 cells. Error bars show standard deviation (three replicates).

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