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. 2019 May 31;294(28):10954–10968. doi: 10.1074/jbc.RA118.007318

Figure 7.

Figure 7.

TRIO directly interacts with RABIN8. A, co-immunoprecipitation of endogenous TRIO and RABIN8 in P21 mouse cerebellum. B, co-immunoprecipitation of EGFP-TRIO8 and FLAG-RABIN8 in Neuro-2a cells. C, co-immunoprecipitation of FLAG-RABIN8 with EGFP-TRIO (1–1295) and TRIO (1296–1909) in COS-7 cells. D, GST-RABIN8 pulldown assay of P21 mouse cerebellum lysates. E, RABIN8 phosphorylation assay to CGNs treated with ITX3. RAC1 activity was decreased. F, quantification of RABIN8 phosphorylation in E. The error bars indicate S.E. (Student's t test); n = 3. G, direct binding assay of His6-RABIN8 to GST–TRIO variants. Coomassie Blue staining of GST–TRIO variants are shown at the bottom. H, COS-7 cells were transfected with FLAG-RABIN8, together with either pEGFP-C3, pEGFP-TRIO(1–230), pEGFP-TRIO(208–673), pEGFP-TRIO(446–909), pEGFP-TRIO(672–1295), or pEGFP-TRIO(1296–1909), and were lysed and subjected to immunoprecipitation. RABIN8 phosphorylation level was determined. I, quantification of the pRABIN8 level in H. The error bars indicate S.E. (one-way ANOVA with Bonferroni's test). *, p < 0.05; **, p < 0.01; n = 6. n.s., not significant; A.U., arbitrary units; IP, immunoprecipitation. MW, molecular weight; WB, Western blot.