Figure 1. Three unbiased criteria to... | Rockefeller University Press

Figure 1.

$Figure 1. Three unbiased criteria to identify CCV proteins. (A–C) Western blots of CCV fractions. (D–F) Corresponding proteomic analyses by SILAC and quantitative mass spectrometry. The ratio of depletion or enrichment (fold change) was calculated for each protein, and proteins were ranked from the highest to the lowest ratio. (A and D) Control CCV fractions compared with mock CCV fractions prepared from clathrin-depleted cells. Genuine CCV proteins (AP-1, AP-2, and CIMPR) are depleted from the mock fraction; contaminants (EF-2) are unchanged. The apparent depletion is more pronounced for AP-1 than for AP-2. (B and E) CCV/cage fractions from auxilin-depleted cells, which accumulate membraneless clathrin cages, compared with control CCV fractions. Endocytic proteins (AP-2 and SNX9) are enriched in the cages, whereas transmembrane cargo proteins (CIMPR) are depleted. (C and F) Improved CCV preparation compared with original preparation. CCV proteins are enriched approximately twofold in the improved CCV fraction (see also Fig. S1). To exclude the possibility that any of the changes were caused by altered transcriptional regulation, we performed a comprehensive microarray gene expression analysis (Fig. S2). Positions of protein molecular mass markers are indicated in kilodaltons in A–C. In some cases, the apparent protein molecular weight is shown instead (indicated by ∼); this was estimated from the average migration of the protein relative to molecular weight markers on at least three gels.$

Three unbiased criteria to identify CCV proteins. (A–C) Western blots of CCV fractions. (D–F) Corresponding proteomic analyses by SILAC and quantitative mass spectrometry. The ratio of depletion or enrichment (fold change) was calculated for each protein, and proteins were ranked from the highest to the lowest ratio. (A and D) Control CCV fractions compared with mock CCV fractions prepared from clathrin-depleted cells. Genuine CCV proteins (AP-1, AP-2, and CIMPR) are depleted from the mock fraction; contaminants (EF-2) are unchanged. The apparent depletion is more pronounced for AP-1 than for AP-2. (B and E) CCV/cage fractions from auxilin-depleted cells, which accumulate membraneless clathrin cages, compared with control CCV fractions. Endocytic proteins (AP-2 and SNX9) are enriched in the cages, whereas transmembrane cargo proteins (CIMPR) are depleted. (C and F) Improved CCV preparation compared with original preparation. CCV proteins are enriched approximately twofold in the improved CCV fraction (see also Fig. S1). To exclude the possibility that any of the changes were caused by altered transcriptional regulation, we performed a comprehensive microarray gene expression analysis (Fig. S2). Positions of protein molecular mass markers are indicated in kilodaltons in A–C. In some cases, the apparent protein molecular weight is shown instead (indicated by ∼); this was estimated from the average migration of the protein relative to molecular weight markers on at least three gels.