JCS -- Brady et al. 121 (20): 3433 Figure IG3

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Fig. 3. Epsin requires CHC to form puncta on the membrane. (A,B) Clathrin and AP2 localization in epsin mutants is comparable to that in the wild type. Confocal images (surface focal planes) of (A) wild-type cells (WT) and (B) epsin-null mutants (epsin^-) expressing clathrin:GFP (green) and immunostained for the ${alpha}$ -adaptin subunit of AP2 ( ${alpha}$ -AP2) (red). (C) Subcellular fractionation of clathrin is similar in wild-type (WT) and epsin-null cells (epsin^-). Immunoblot of samples probed with ${alpha}$ -CHC antibodies. Lys, lysate; LSP, low-speed (3000 g) pellet; LSS, low-speed supernatant; HSP, high-speed (100,000 g) pellet; HSS, high-speed supernatant. (D) Epsin localizes to the membrane but does not form puncta in CHC-null cells. Confocal image (middle focal plane) of CHC-null cells (CHC^-) expressing epsin:GFP (E) Subcellular fractionation of epsin is altered in CHC mutants, but not in ${alpha}$ -adaptin-null mutants. Immunoblot probed with anti-epsin antibodies. (F) Epsin forms puncta that colocalize with AP2 on the membranes of CLC mutants. Confocal images (surface focal plane) of CLC-null mutants expressing epsin:GFP (green) and immunostained for the ${alpha}$ -adaptin subunit of AP2 ( ${alpha}$ -AP2) (red). (G) Epsin forms reduced numbers of puncta at the plasma membrane in ${alpha}$ -adaptin-null mutants that colocalize with clathrin. Confocal images (surface focal plane) ${alpha}$ -adaptin mutants (AP2^-) expressing epsin:GFP (green) and immunostained for clathrin (red). Scale bars, 5 µm.