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 .