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. 2019 Feb 25;20(4):e46794. doi: 10.15252/embr.201846794

Figure 2. BAG6 physically interacts with Rab8a in a nucleotide‐binding specific manner.

Figure 2

  1. Protein interaction network suggested by public databases.
  2. BAG6 protein co‐precipitated Rab8a, while neither Rab7 nor Rab11a was co‐precipitated with BAG6. Flag‐tagged Rab8a, Rab7, Rab11a, and luciferase‐CL1 (Lc‐CL1; a positive control) were expressed in HeLa cells and the cells were treated with 10 μM MG‐132 for 4 h. Flag‐immunoprecipitates were blotted with anti‐BAG6 and anti‐Flag antibodies, respectively. Note that all cells used were treated with 10 μM MG‐132 for 4 h.
  3. S‐tagged BAG6 pull‐down efficiently co‐precipitated Rab8a (T22N), a GDP‐bound mutant, while BAG6 scarcely co‐precipitated Rab8a (Q67L), a constitutively active mutant. Co‐precipitation of Rab8a WT with BAG6 was used as a standard. MG‐132 (10 μM) was included in the cell culture for 4 h, as indicated. Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L, and that this was partly due to increased degradation, as will be shown later. S‐BAG6 stands for N‐terminally S‐tagged BAG6 protein (see the Materials and Methods).
  4. Anti‐Flag signals in (C) were quantified, and relative signal intensities are presented. The value of the WT Rab8a signal with MG‐132 was defined as 1.0. Note that all signal intensities of Flag‐tag were normalized by that of the Rab8a input signals. The graph represents the mean ± standard deviation (SD) calculated from three independent biological replicates. An asterisk indicates P < 0.05 (Student's t‐test).
  5. A series of Flag‐Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were co‐precipitated with Flag‐Rab8a. Note that all cells used were treated with 10 μM MG‐132 for 4 h.
  6. Deficiency of Rabin8, a GEF for Rab8a, enhanced the physical interaction between BAG6 and WT Rab8a proteins. Flag‐tagged WT Rab8a was expressed in Rabin8 siRNA‐treated cells, and Flag‐immunoprecipitates were probed with an anti‐BAG6 antibody. Note that all cells used were treated with 10 μM MG‐132 for 4 h.

Source data are available online for this figure.