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. 2016 Mar 2;6:22539. doi: 10.1038/srep22539

Figure 4. Hypersecretion phenotype of the Rab37-knockdown RBL-2H3 cells requires an intact Rab27-Munc13-4 system.

Figure 4

(A,B) SiRNA-mediated downregulation of the Rab27 subfamily proteins (A) and Munc13-4 (B). Cell lysates (20 μg) from the G418-surviving cells transfected with Si-control plasmid together with Rab27a-specific siRNA (Si-Rab27a), Rab27b-specific siRNA (Si-Rab27b), Munc13-4-specific siRNA (Si-Munc13-4), or control siRNA (Si-control) were subjected to western blot analyses with the antibodies indicated. Arrowheads indicate position of the Rab27 subfamily proteins (A). Full-length blotting images of (A,B) are presented in Supplementary Figs. S10 and S12, respectively. The graphs represent the relative quantity of the Rab27 subfamily proteins (A) and Munc13-4 (B) with the quantity in the Si-control transfectant set to 100. Bars represent mean ± SD (n = 4). ***p < 0.001; **p < 0.01; NS, not significant. (C) Effects of Rab27 knockdown on antigen-induced degranulation. G418-surviving cells transfected with Si-control plasmid together with Si-control, Si-Rab27a, Si-Rab27b, or a combination of Si-Rab27a and Si-Rab27b (Si-Rab27a/b) were used. (D,E) Effect of Rab27 (D) or Munc13-4 (E) knockdown on antigen-induced degranulation in Rab37-knockdown cells. G418-surviving cells transfected with the following combinations were used: Si-control plasmid plus Si-control (Si-control), Si-Rab37 plasmid plus Si-control (Si-Rab37), Si-control plasmid plus Si-Rab27a/b (Si-Rab27a/b), Si-Rab37 plasmid plus Si-Rab27a/b (Si-Rab37 + Si-Rab27a/b), Si-control plasmid plus Si-Munc13-4 (Si-Munc13-4), or the Si-Rab37 plasmid plus Si-Munc13-4 (Si-Rab37 + Si-Munc13-4). In (C−E) the cells were sensitised and stimulated with or without antigen for 10 min. The degree of β-hexosaminidase release was measured as in Fig. 1(C−F) and the release without stimulation was subtracted. Error bars represent mean ± SD (n = 4). *p < 0.05; **p < 0.01; NS, not significant.