anti--tubulin mouse monoclonal antibody ( sigma - aldrich corp ) , anti - arf6 mouse monoclonal antibody ( santa cruz biotechnology , inc ) , anti - rab11 rabbit polyclonal antibody , anti - tfr mouse monoclonal antibody ( invitrogen corp ) , anti - eea-1 mouse monoclonal antibody and anti - gm130 mouse monoclonal antibody ( bd biosciences ) were obtained commercially .
s2 was performed with a fluorophore - conjugated anti - tfr antibody that was generated by using the zenon antibody labeling kit ( invitrogen corp ) . using this antibody
slightly enhanced the tfr signals at the plasma membrane ( figs . s1 and s2 ) .
all of the procedures used to perform cell culture , immunoblotting and the immunofluorescence analyses have been described elsewhere .
anti--tubulin mouse monoclonal antibody ( sigma - aldrich corp ) , anti - arf6 mouse monoclonal antibody ( santa cruz biotechnology , inc ) , anti - rab11 rabbit polyclonal antibody , anti - tfr mouse monoclonal antibody ( invitrogen corp ) , anti - eea-1 mouse monoclonal antibody and anti - gm130 mouse monoclonal antibody ( bd biosciences ) were obtained commercially .
s2 was performed with a fluorophore - conjugated anti - tfr antibody that was generated by using the zenon antibody labeling kit ( invitrogen corp ) . using this antibody
slightly enhanced the tfr signals at the plasma membrane ( figs . s1 and s2 ) .
all of the procedures used to perform cell culture , immunoblotting and the immunofluorescence analyses have been described elsewhere .
 recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane . to determine how recycling endosomes perform their functions , searching for proteins and lipids that specifically localized at recycling endosomes 
 has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers . 
 however , it remains unclear whether all the conventional markers have identical localizations . 
 here we report finding that three well - known recycling endosome markers , i.e. , arf6 , rab11 and transferrin receptor ( tfr ) , have different intracellular localizations in pc12 cells . 
 the results of immunofluorescence analyses showed that the signals of endogenous arf6 , rab11 and tfr in nerve growth factor - stimulated pc12 cells generally differed , although there was some overlapping . 
 our findings provide new information about recycling endosome markers , and they highlight the heterogeneity of recycling endosomes . 
Materials and Methods
Antibodies
Cell culture, immunoblotting and immunofluorescence analyses
Supplementary Material