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Fig. 7. Subcellular distribution of transfected mutated or wild-type PTPL1-FERM domain or full-length PTPL1. Transfected COS1 cells were fractionated and the resulting fractions were analyzed on a 10% SDS/PAGE and immunoblotted. F-actin and intermediate filaments containing vimentin were recovered in the particulate fraction (P, pellet), whereas G-actin and depolymerised tubulin were recovered in the soluble fraction (S, supernatant). The equal amount of proteins loaded in each fraction was checked by Coomassie blue staining. (A) Transfection of FERM domains. Transfected HA-epitope tagged wild-type FERM domain was found to be strongly associated with the P fraction as detected after immunoblotting with anti-HA monoclonal antibody. KN1-FERM mutant displayed an equivalent distribution. KN2-and KN1-2-FERM mutants were only detected in the S fraction. (B) Transfection of full-length PTPL1. Transfected HA-epitope tagged PTPL1 was mainly recovered in the S fraction and significantly in the P fraction. KN1-2 or {Delta}FERM mutants were only detected in the S fraction. To ensure proper fractionation, membranes were immunoblotted with anti-actin polyclonal antibody, anti-vimentin and anti-tubulin monoclonal antibodies.