Figure 8. Capture of AP-1 vesicles using... | Rockefeller University Press

Figure 8.

$Capture of AP-1 vesicles using magnetic beads. (A) A CCV-enriched fraction was prepared from HeLa cells expressing mRuby2-tagged AP-1 γ, and then the AP-1-positive vesicles were captured just before the final centrifugation step using a rabbit antiserum against mRuby2 followed by protein A-coated magnetic beads. CCVs (arrowheads) can be seen to be associated with the beads; the two top ones are shown magnified in the inset. Scale bars: 100 nm. (B) Coomassie blue-stained gel of the whole cell homogenate, proteins captured on magnetic beads, and the CCV-enriched fraction from cells expressing mRuby2-tagged AP-1 γ. As a control for specificity, the preimmune serum from the same rabbit was also incubated with protein A beads. The samples containing beads and the CCV-enriched fraction were made up to the same volume so the efficiency of capture could be assessed, and the sample containing cell homogenate was made up to the same protein concentration as the sample containing the CCV fraction. H and L refer to immunoglobulin heavy and light chains; the bands labeled 1–4 were excised and found to correspond to CIMPR, CHC17, the AP-1 γ subunit, and the AP-1 β1 subunit, respectively. (C) Western blot of the samples shown in B probed with various antibodies. The dotted lines indicate the position on the gel for each antigen. In addition to the expected proteins, a small amount of AP-2 was also captured by the beads. GGA2 has a similar profile to AP-2, with relatively little captured by the beads. Source data are available for this figure: SourceData F8.$

Capture of AP-1 vesicles using magnetic beads. (A) A CCV-enriched fraction was prepared from HeLa cells expressing mRuby2-tagged AP-1 γ, and then the AP-1-positive vesicles were captured just before the final centrifugation step using a rabbit antiserum against mRuby2 followed by protein A-coated magnetic beads. CCVs (arrowheads) can be seen to be associated with the beads; the two top ones are shown magnified in the inset. Scale bars: 100 nm. (B) Coomassie blue-stained gel of the whole cell homogenate, proteins captured on magnetic beads, and the CCV-enriched fraction from cells expressing mRuby2-tagged AP-1 γ. As a control for specificity, the preimmune serum from the same rabbit was also incubated with protein A beads. The samples containing beads and the CCV-enriched fraction were made up to the same volume so the efficiency of capture could be assessed, and the sample containing cell homogenate was made up to the same protein concentration as the sample containing the CCV fraction. H and L refer to immunoglobulin heavy and light chains; the bands labeled 1–4 were excised and found to correspond to CIMPR, CHC17, the AP-1 γ subunit, and the AP-1 β1 subunit, respectively. (C) Western blot of the samples shown in B probed with various antibodies. The dotted lines indicate the position on the gel for each antigen. In addition to the expected proteins, a small amount of AP-2 was also captured by the beads. GGA2 has a similar profile to AP-2, with relatively little captured by the beads. Source data are available for this figure: SourceData F8.