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The p11/S100A10 Light Chain of Annexin A2 Is Dispensable for Annexin A2 Association to Endosomes and Functions in Endosomal Transport
Figure 3
Biochemical analysis of p11 distribution and annexin A2/p11 interaction detection.
(A), Total cell lysates and early endosomes (EE) were prepared from BHK cells, and then analyzed by SDS gels (equal proteins amounts loaded in each lane) and western blotting using the indicated antibodies. The p11 and AnxA2 signals were scanned and the right panel shows the ratio of the p11 over AnxA2 signals in the corresponding fractions (A.U: arbitrary units). (B) Experiments and quantification (right panel) were as in (A) except that total membranes and cytosol obtained after high speed centrifugation were analyzed. (C) Hela cells were transfected with (AnxA2H28-GFP), and cell lysates were prepared. Then, AnxA2-GFP was immunoprecipitated from the lysates with anti-GFP antibodies (IP: immunoprecipitate; Ab- : control without the specific antibody; load: input fraction before immunoprecipitation). Samples were then analyzed by SDS gels and western blotting with the indicated antibodies. The H28 antibody only recognizes AnxA2H28, while, in our hands, the HH7 antibody recognizes WT endogenous AnxA2. HC IgG, heavy chain of anti-GFP antibody used for immunoprecipitation. (D) The experiments were as in (C), except that purified early endosomes were used as starting materials. The left panel shows western blots with anti-GFP antibody, while the right panels show blots with anti-AnxA2 antibody. For comparison, the small right panel (EE load) indicates the mobility of (untagged) AnxA2 in a gel of the early endosome (EE) starting material (load). HC IgG, heavy chain of anti-GFP antibody used for immunoprecipitation, LC IgG, light chain of anti-GFP antibody.