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. 2017 Mar 6;114(11):E2176–E2185. doi: 10.1073/pnas.1617981114

Fig. 3.

Fig. 3.

Endogenous STX11 interacts with SNAP23 and VAMP8 in activated CTLs. (A) Normal human donor CTLs purified from peripheral blood were activated using beads coated with anti-CD3 and -CD28 antibodies for 4 h. Endogenous STX11 and STX5 were immunoprecipitated from cell lysates using either rabbit anti-STX11 or rabbit anti-STX5 antibody and the indicated coimmunoprecipitated SNAREs or Munc18-2 were analyzed by Western blotting. (B) Bands in the Western blot that corresponded to the fraction of VAMP4/7/8 that coprecipitated with either STX11 or STX5 were quantified by densitometry and normalized to the total amount of STX11 or STX5, respectively, and immunoprecipitated in the same lane. Results represent mean ± SD of three independent experiments. (C) Endogenous VAMP8 was immunoprecipitated from activated CTL lysates as in A, and the indicated coimmunoprecipitated SNAREs or Munc18-2 was analyzed by Western blotting. Note that a goat anti–Munc18-2 antibody was used for Western blot in A, D, and E, giving a single band, whereas a rabbit anti–Munc18-2 antibody was used in C, giving a doublet. (D and E) HeLa cells were transiently transfected with HA–STX11. Immunoprecipitation from cell lysates was performed using either anti-HA (D) or IgG control antibody (E), and coimmunoprecipitated VAMP8, SNAP23, and Munc18-2 were analyzed by Western blotting. Blots are representative of three experiments.