Changes in protein spot expression in response to UPF1 knockdown. A. Representative western blot of 10 μg total cell protein for UPF1 and actin as a loading control for each sample. C2 treated sample was diluted 1:2, 1:4 and 1:8 in ASB14 buffer in order to estimate the degree of knockdown. B. Representative U2AF35 RT-PCR assay on parallel RNA samples. The adjoining cartoon illustrates the identity of each band. The lower band results from alternative inclusion of one of a pair of normally mutually exclusive exons of equal size, termed E3 (yielding isoform U2AF35a) and EAb (yielding isoform U2AF35b) . The upper band represents inclusion of both exons, which results in a frameshift that creates a PTC - making isoform U2AF35c NMD sensitive . M: 1 kb plus marker (GE healthcare). RT-: addition of RT performed without reverse transcriptase. PCR-: PCR performed without template. Underlying numbers indicate the percentage of the signal from both bands represented by he upper, double-included NMD-sensitive, band for each siRNA treatment (mean of 6 biological replicates ± SEM). C. Proportional Venn diagrams representing the number of protein spot changes unique and common to each siRNA against UPF1. The upper, red coloured, diagram details upward changes whereas the lower, blue coloured diagram details downward changes. B. Principal component analysis (PCA) scores plot illustrating the similarity of the multi-gel study samples to each other by their relationships with the first two principal components (PCs) describing the whole multi-gel study dataset. Blue squares - C2 treated samples; red circles - Upf1_A treated samples, green triangles - Upf1_B treated samples. t - score relating to PC1; t - score relating to PC2.