Verification of novel CLIC cargo. (A) NIH3T3 cells were incubated with either anti–Thy-1 or anti-CD44 antibodies and CTxB-555 and Tf-647 for 2 min. Myoferlin was detected after fixation, showing steady-state localization. Arrows indicate colocalization. Bar, 10 µm. (B) 3T3-GPI cells were left untreated or were treated with 100 nM wortmannin before internalization of anti-GFP for 10 min. Arrows indicate colocalization. Bar, 10 µm. (C) Quantitation of B from 12–15 cells in three independent experiments. (D) Quantitation of colocalization between internalized anti-CD44 antibodies and Tf-647, CTxB-555, or anti-myc antibodies for GFP-dysferlin-myc–expressing cells after 2, 10, and 40 min. Error bars show SEM. (E) Anti–CD44-HRP was internalized into WT MEFs before DAB reaction. Arrows show anti-CD44-HRP–positive carriers with morphology of CLICs. Arrowheads show large, tubular ring-shaped anti-CD44-HRP–positive compartment. Bars, 200 nm. (F) Anti–CD44-HRP or Tf-HRP was pulsed into Cav1^−/− MEFs for 15 s, 1 min, or 2 min. Cells were fixed and processed for vertical sections. Arrows show HRP-labeled carriers. Bar, 200 nm. (G) Stereology measurements were captured across 20–25 cells in three independent areas as treated in F. Error bars show SEM.