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Fig. 9. Effect of neomycin treatment on subcellular distribution of PTPL1. (A) Transfection of ezrin-FERM domain. COS1 cells were transfected with VSV-G-epitope tagged N-terminal part of ezrin (FERM domain, 1-310 aa) and were fractionated after treatment with or without 10 mM neomycin for 24 hours. Association of ezrin-FERM domain with particulate fraction was strongly inhibited by such cell treatment. (B) 35S-labeled PTPL1-FERM domain was incubated in TBST 3% BSA with PIP-Strips (Echelon Biosciences) pre-saturated with TBST 3% BSA [C] or TBST 3% BSA containing 1 mM neomycin [Neo]. (C) Transfection of PTPL1-FERM domains. COS1 cells were transfected with either HA-epitope tagged wild-type FERM domain or KN1-2-FERM domain, treated or not with neomycin as described above. Similarly to ezrin-FERM domain, association of wild-type FERM domain with P fraction was impaired by neomycin treatment. On the contrary, the distribution of the KN1-2-FERM mutant was not affected by such a treatment. (D) Transfection of full-length PTPL1. Similarly, COS1 cells were transfected with HA-epitope tagged wild-type PTPL1, PTPL1 KN1-2 or PTPL1 {Delta}FERM, and fractionated after neomycin treatment. Neomycin treatment strongly inhibited the association of wild-type PTPL1 with the P fraction. Moreover, KN1-2 and {Delta}FERM mutant distributions were not affected.