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. Author manuscript; available in PMC: 2018 Sep 13.
Published in final edited form as: Cell Host Microbe. 2017 Sep 13;22(3):330–342.e4. doi: 10.1016/j.chom.2017.08.002

Figure 6. Co-dependency of the RAB11a and RAB27b-associated trafficking circuits for efficient bacterial expulsion.

Figure 6

(A) Rab27b-binding domain (R27BD) from the RAB27b effector SLAC2 was cloned and fused to GST. The purified R27BD-GST protein was immobilized and used to pulled down GTP-bound active RAB27b from uninfected or infected BECs transfected with control siRNA or siRNA targeting RAB11a. The proteins of interest in the total cell lysates (TCL) were depicted to demonstrate that similar levels of RAB27b protein were present in each fraction.\

(B) RAB27b immunoprecipitated from uninfected or infected BECs transfected with control siRNA or siRNA targeting RAB11a. The association between RAB27b with its effector MyosinVIIa in RAB11a-expressing or deficient cells was examined by western blotting of the IP fractions. The proteins of interest in the total cell lysates (TCL) were depicted to demonstrate that similar levels of MyosinVIIa protein were present in each fraction.

(C) Imunoblots of MyRIP or MyosinVIIa in total cell lysates (TCL) or bacteria-containing vesicle (BCV) fractions isolated from infected BECs which are pre-transfected with control siRNA or siRNA targeting RAB11a.

(D) Rab11-binding domain (R11BD) from the RAB11a effector RAB11FIP3 was cloned and fused to GST. The purified R11BD-GST protein was used to pulled down GTP-bound active Rab11a from uninfected or infected BECs transfected with control siRNA or siRNA targeting RAB27b or RAB27b effector MyRIP or MyosinVIIa. The proteins of interest in the total cell lysates (TCL) were depicted to demonstrate that similar levels of RAB11a protein were present in each fraction.

(E) Immunoprecipitation of RAB11a from uninfected or infected BECs transfected with control siRNA or siRNA targeting RAB27b or its effector MyRIP or MyosinVIIa. The association between RAB11a with its effector RAB11FIP3 in control or KD cells were examined by western blotting of the IP fractions. The proteins of interests in the total cell lysates (TCL) were depicted to demonstrate that similar levels of RAB11FIP3 protein were present in each fraction.

(F) Immunoblots of RAB11FIP3 in total cell lysates (TCL) or bacteria-containing vesicle (BCV) fractions isolated from infected BECs which are pre-transfected with control siRNA or siRNA targeting RAB27b or its effector MyRIP or MyosinVIIa.

(G) RAB27b immunoprecipitated from naïve or infected BECs transfected with control siRNA or siRNA targeting RAB11a. The association between RAB27b with Exocyst complex protein SEC15 in RAB11a expressing or deficient cells were examined by western blotting of the IP fractions. The proteins of interest in the total cell lysates (TCL) were depicted to demonstrate that similar levels of SEC15 protein were present in each fraction.

(H) RAB11a was immunoprecipitated from uninfected or infected BECs transfected with control siRNA or siRNA targeting RAB27b. The association of RAB11a with Exocyst complex protein SEC15 in control or KD cells was examined by western blotting of the IP fractions. The proteins of interest in the total cell lysates (TCL) were depicted to demonstrate that similar levels of SEC15 protein were present in each fraction.