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. 2016 Aug 30;11(8):e0162050. doi: 10.1371/journal.pone.0162050

Fig 3. Extraction of clathrin from synaptosome lysate by GST-tagged WT, Site 1 and 2 mutated AP180 and CALM.

Fig 3

(A) AP180 and (B) CALM mutated at Site 1, Site 2 and Site 1&2 (double mutant) were used in 1 h pull-downs with rat synaptosome lysate and the amount of clathrin bound was determined by Western blot with anti-clathrin heavy chain (CHC) using 40% of the sample (representative blot shown from two 1 h pull-down experiments). Ten percent of the sample was resolved by SDS-PAGE and stained with Coomassie for comparison of bait levels. The experiment was performed twice more, except the pull-down was for 10 min instead of 1 h and the result was very similar (Figure B in S1 File). (C) and (D) Comparison of clathrin binding from the combined densitometry of 10 min and 1 h pull-downs with the AP180 mutants normalised to AP180 WT and the CALM mutants to CALM WT. Data is expressed as average relative amount of clathrin bound as a fraction of the WT pull-down ± SEM (n = 4; ***, P < 0.001 compared to WT; ns = not significant).