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. 2012 Aug 30;7(8):e44252. doi: 10.1371/journal.pone.0044252

Figure 1. The carboxy-terminal 69 amino acids of PICALM are essential for binding clathrin and interfering with TfR endocytosis.

Figure 1

(A) Schematic representation of amino-terminal-tagged GFP-PICALM fusion proteins. (B) TfR internalization in HEK293 cells transiently transfected with empty vector (control), full length PICALM (PICALM: 1–652), amino-terminal truncated PICALM (PICALM: 256–652), or a PICALM mutant missing both the amino and carboxy-terminal regions (PICALM: 256–583). The relative rate of endocytosis was calculated as described in Materials and Methods. Nexp  = 11. *p<0.001 compared with control. (C) Western blot of proteins co-immunoprecipitated (IP) with anti-GFP antibody and immunoblotted (IB) with anti-clathrin heavy chain (CHC) antibody (upper panel). Cell lysates (input) were also immunoblotted with anti-CHC and anti-PICALM antibodies (lower panels). The molecular weights of GFP-PICALM, GFP-PICALM:256–652 and GFP-PICALM:256–583 are 97kD, 70kD and 62 kD, respectively.