Fig. 8

Sec14l3/SEC14L2 promote RAB5A/4A activation. a Sec14l3 interacts preferentially with GDP-bound state of RAB4A/5 A. HEK293T cells were transfected with Sec14l3-GFP and lysed for in vitro incubation with GTPγS/GDP loaded-RAB4A/5 A respectively. Quantification data of the relative protein levels of bound Sec14l3 are shown on the right (n = 3). *p < 0.05. b Recombinant Sec14l3 protein purified from E. coli binds preferentially to GDP-bound RAB4A/5 A in vitro. Recombinant GST-Sec14l3 protein purified from E. coli was loaded with GTPγS and immobilized for incubation with the same amount of GTPγS/GDP loaded-RAB4A/5A. Quantification data of the relative protein levels of bound RAB4A/5A are shown on the right (n = 3). *p < 0.05. c Knockdown of SEC14L2 alleviates RAB5A-GTP levels in HEK293T cells. NC or SEC14L2 shRNA transfected cell lysates were immunoprecipitated by an anti-RAB5A-GTP antibody or subjected to GST-R5BD pull down. Quantification of the relative protein level of RAB5A-GTP is shown on the right (n = 3). **p < 0.01, *p < 0.05. d Knockdown of SEC14L2 alleviates RAB4A-GTP level in HEK293T cells. NC- or SEC14L2 shRNA transfected HEK293T cell lysates were immunoprecipitated by anti-RAB4A-GTP antibody and quantification of the relative protein level of RAB4A-GTP is shown on the right (n = 3). *p < 0.05. t-tests were used for statistical analyses in a–d. e Proposed working model of Sec14l3/SEC14L2 for RAB5A/4A-mediated VEGFR2 trafficking. Upon ligand stimulation, VEGFR2 could be internalized and recycled through RAB5A and RAB4A mediated pathways. Sec14l3/SEC14L2 are beneficial for increasing RAB5A/4A in their GTP-bound forms to accelerate the movement of VEGFR2-containing vesicles to early endosomes and subsequently recycling endosomes, consequently preventing VEGFR2 from PTP1B-mediated dephosphorylation