Extended Data Fig. 10: Phosphorylation at Thr75 promotes Rab1A interacting with ER-shaping proteins.
a, Left, anti-mCherry immunoprecipitates were prepared from MDA-MB-231 cells expressing mCherry-tagged wild-type or mutant Rab1A and analyzed by immunoblotting. Right, co-immunoprecipitated RTN4B was quantified. b, Left, anti-GFP immunoprecipitates were prepared from MDA-MB-231 cells expressing GFP-tagged wild-type or mutant Rab1A and analyzed by immunoblotting. Right, co-immunoprecipitated ATL3 was quantified. c, HA-ATL3 was bound to anti-HA magnetic beads and then incubated with purified GST-Flag-Rab1A (wild-type or mutant). The samples were analyzed by immunoblotting. d, GST-tagged wild-type or mutant Rab1A immobilized to glutathione Sepharose 4B was used for the binding assay with the COS-7 cell lysates and the samples analyzed by immunoblotting. e, mCherry-ATL3 was co-expressed with Flag-tagged WT Rab1A, Rab1A-T75A, or Rab1A-T75D in MDA-MB-231 cells. Anti-mCherry immunoprecipitation was performed and analyzed by immunoblotting. f, Left, cell lysates from COS-7 cells overexpressing RTN4B alone or co-expressing RTN4B and Rab1A (wild-type or mutant) were treated with the indicated concentrations of EGS and analyzed by immunoblotting. *, monomer; **, dimer. Right, RTN4B dimers were quantified. g, Left, cell lysates from COS-7 cells expressing wild-type or mutant Rab1A were treated with the indicated concentrations of EGS and analyzed by immunoblotting with anti-Reep5 antibody. *, monomer; **, dimer; ***, trimer. Right, Reep5 dimers were quantified. h, Left, cell lysates from COS-7 cells overexpressing Reep5 alone or co-expressing Reep5 and Rab1A (wild-type or mutant) were treated with the indicated concentrations of EGS and analyzed by immunoblotting. *, monomer; **, dimer; ***, trimer. Right, Reep5 dimers were quantified. Data shown in a-h are representative of three independent experiments. Data from a, b were analyzed using two-tailed unpaired Student’s t-test. Data are presented as mean ± s.e.m.
