hela cells were cultured in dmem ( media tech ) with 10% fetal bovine serum ( hyclone ) and 1% penicillin streptomycin ( invitrogen ) .
hela cells stably expressing m4-blast constructs were established by lentivirus infection followed by selection with 4 g / ml blasticidin ( invitrogen ) .
rin1 wild type , rin1 were made in in pm4-blast vector . in rin1 , tyrosines 36 , 121 , 148 , and 295 were mutated to phenyalanine .
the rab5-gtp pulldown construct was created using the zn finger domain of rabenosyn ( zfyve20 ) , a rab5 binding domain .
the sequence encoding amino acids 140 of human zfyve20 was amplified using forward primer 5-atgcgctagcagatctactagtatggcttctctggacgaccc and reverse primer 5-atgcggatccttatctagattcccctgagtgttcttcct .
ligation compatible restriction sites in each primer ( xbai / spei and bamhi / bglii ) were used for stepwise head - to - tail additions in pks bluescript .
the 4x concatemer was then inserted into the bamhi - ecori sites of pgex-2 t to create pgex-4xzfyve ( r5bd - gst ) .
active rab5 from cells were pulled down using the construct : pgex-4xzfyve - gst , purified on glutathione beads as previously described . briefly
cells were serum starved for about 18 h. stimulation was performed with control medium or 100 ng / ml egf medium .
cells were lysed after the respective times with np-40 lysis buffer ( 150 mm tris ph 8.0 , 50 mm nacl , 1% np-40 ) containing 1 mm pmsf , 10 ug / ml leupeptin , 1 um pepstatin and 1 mm sodium orthovanadate ( sigma aldrich ) in the presence of 10 mm mgcl2 .
lysates were subject to pull - down at 4 c with 4x - zfyve - gst on glutathione beads in order to enrich for activated rab5 .
beads were then washed with the lysis buffer , boiled in laemmli loading buffer , followed by gel electrophoresis and analysis of rab5 by immunoblotting . for immunoblotting ,
the membranes were blocked with 5% milk in tbst ( 0.1% tween-20 ) followed by incubation with primary and secondary antibodies at room temperature .
the membranes were washed with tbst between the incubations and developed using the ecl plus western blotting reagent ( vwr ) or scanned using a li - cor odyssey scanner .
quantification of immunoblots was performed using the li - cor odyssey software or imagej ( nih ) .
antibodies used for immunoblotting and their sources were pan - rab5 1:1000 ( abcam , # ab18211 ) , tubulin 1:5000 ( sigma aldrich , # t6074 - 200 ul ) , rin1 1:1000 ( mouse mab , clone # c9e11 , colicelli lab , abpro ) , sheep - anti - mouse - hrp 1:3000 ( amersham biosciences , # na931 ) , goat - anti - rabbit - hrp 1:3000 ( kirkegaard and perry , # 4741506 ) , goat - anti - rabbit - irdye 800 1:5000 ( li - cor biosciences , # 926 - 32211 ) and goat - anti - mouse - irdye 680 1:5000 ( li - cor biosciences , # 926 - 32220 ) .
cells were serum starved for about 12 h. boyden chambers ( cell culture inserts , 8 m , bd falcon ) were coated with 10 ug / ml fibronectin overnight at 4 c .
the cell culture inserts were rinsed once in pbs and placed on 24-well plates , containing either control medium or 100 ng / ml egf medium .
cells were allowed to migrate across the transwell at 37 c for 24 h. the chambers were then rinsed in pbs and fixed with 4% pfa .
cells that had migrated were stained with crystal violet and counted under the microscope using a hemocytometer .
hela cells were cultured in dmem ( media tech ) with 10% fetal bovine serum ( hyclone ) and 1% penicillin streptomycin ( invitrogen ) .
hela cells stably expressing m4-blast constructs were established by lentivirus infection followed by selection with 4 g / ml blasticidin ( invitrogen ) .
all rin1 expression constructs were made in lentivirus vectors . rin1 wild type , rin1 were made in in pm4-blast vector . in rin1 , tyrosines 36 , 121 , 148 , and 295 were mutated to phenyalanine .
the rab5-gtp pulldown construct was created using the zn finger domain of rabenosyn ( zfyve20 ) , a rab5 binding domain .
the sequence encoding amino acids 140 of human zfyve20 was amplified using forward primer 5-atgcgctagcagatctactagtatggcttctctggacgaccc and reverse primer 5-atgcggatccttatctagattcccctgagtgttcttcct .
ligation compatible restriction sites in each primer ( xbai / spei and bamhi / bglii ) were used for stepwise head - to - tail additions in pks bluescript .
the 4x concatemer was then inserted into the bamhi - ecori sites of pgex-2 t to create pgex-4xzfyve ( r5bd - gst ) .
active rab5 from cells were pulled down using the construct : pgex-4xzfyve - gst , purified on glutathione beads as previously described . briefly
cells were serum starved for about 18 h. stimulation was performed with control medium or 100 ng / ml egf medium .
cells were lysed after the respective times with np-40 lysis buffer ( 150 mm tris ph 8.0 , 50 mm nacl , 1% np-40 ) containing 1 mm pmsf , 10 ug / ml leupeptin , 1 um pepstatin and 1 mm sodium orthovanadate ( sigma aldrich ) in the presence of 10 mm mgcl2 .
lysates were subject to pull - down at 4 c with 4x - zfyve - gst on glutathione beads in order to enrich for activated rab5 .
beads were then washed with the lysis buffer , boiled in laemmli loading buffer , followed by gel electrophoresis and analysis of rab5 by immunoblotting . for immunoblotting ,
the membranes were blocked with 5% milk in tbst ( 0.1% tween-20 ) followed by incubation with primary and secondary antibodies at room temperature .
the membranes were washed with tbst between the incubations and developed using the ecl plus western blotting reagent ( vwr ) or scanned using a li - cor odyssey scanner .
quantification of immunoblots was performed using the li - cor odyssey software or imagej ( nih ) .
antibodies used for immunoblotting and their sources were pan - rab5 1:1000 ( abcam , # ab18211 ) , tubulin 1:5000 ( sigma aldrich , # t6074 - 200 ul ) , rin1 1:1000 ( mouse mab , clone # c9e11 , colicelli lab , abpro ) , sheep - anti - mouse - hrp 1:3000 ( amersham biosciences , # na931 ) , goat - anti - rabbit - hrp 1:3000 ( kirkegaard and perry , # 4741506 ) , goat - anti - rabbit - irdye 800 1:5000 ( li - cor biosciences , # 926 - 32211 ) and goat - anti - mouse - irdye 680 1:5000 ( li - cor biosciences , # 926 - 32220 ) .
cells were serum starved for about 12 h. boyden chambers ( cell culture inserts , 8 m , bd falcon ) were coated with 10 ug / ml fibronectin overnight at 4 c .
the cell culture inserts were rinsed once in pbs and placed on 24-well plates , containing either control medium or 100 ng / ml egf medium .
cells were allowed to migrate across the transwell at 37 c for 24 h. the chambers were then rinsed in pbs and fixed with 4% pfa .
cells that had migrated were stained with crystal violet and counted under the microscope using a hemocytometer .
 stimulation of a receptor tyrosine kinase ( rtk ) , such as egfr , leads to ras activation followed by rin1 activation . 
 rin1 , in turn , activates rab5 family gtpases , as well as abl tyrosine kinases . 
 as expected , rin1 expression directly correlates with rab5-mediated egfr endocytosis . 
 we previously showed that normal receptor endocytosis and internalized egfr fate also depend on the ability of rin1 to concomitantly activate abl tyrosine kinases , consistent with the established role of abl kinases in cytoskeleton remodeling and the growing evidence that such remodeling plays a role in endocytic processes . here 
 we report that growth factor - directed cell migration , a physiological process that involves receptor endocytosis and actin remodeling , also requires the ability of rin1 to coordinate rab5 gtpase and abl tyrosine kinase pathways . 
Materials and Methods
Cell culture and reagents
Expression constructs
RAB5 pull-down
Cell migration