Figure 6
Expression of C1QTNF5 in hTERT-RPE1 cells. (a) Recombinant C1QTNF5 is a 28 kDa monomer in reduced and denatured transiently transfected hTERT-RPE1 cell lysates, immunoblotted for C1QTNF5, with beta-actin as a loading control. (b) densitometric analysis shows that mutant proteins are expressed significantly less than WT C1QTNF5 intracellularly, after normalisation to beta-actin. *p < 0.05,**p < 0.01, ***p < 0.001 (T-test). (c) Secreted C1QTNF5 is a monomer of 28 kDa. Mutant C1QTNF5 shows varying levels of secretion, quantified by densitometry in (d). (e) Secreted C1QTNF5 forms complexes, including trimers and higher order structures, that are electrophoretically stable under non-reducing conditions (f) The high molecular weight complex of C1QTNF5 is held together by disulphide bonds and is not observed under reducing conditions. Complex formation observed in (e) is disrupted by the pathogenic mutations, quantified by densiotometry in (g). Statistically significant differences in the proportion of total C1QTNF5 that forms stable HMW complexes are indicated. (h) Mutation of C1QTNF5 results in decreased apical secretion and a shift towards a greater proportion of C1QTNF5 secreted from the basal surface of transiently transfected cells grown on a transwell membrane, quantified by densitometry in (i). Representative blots from a minimum of three separate transfections are shown, error bars show standard error of mean (s.e.m.). Full-length blots are presented in Supplementary Figure 3.
