Skip to main content
. 2016 Mar 2;6:22539. doi: 10.1038/srep22539

Figure 2. Hypersecretion phenotype of Rab37-knockdown RBL-2H3 cells.

Figure 2

(A) Rab37 mRNA expression in the Si-Rab37 transfectant. The relative mRNA expression of Rab37 compared to GAPDH was determined in the G418-surviving cells transfected with Si-Rab37 or Si-control plasmid using quantitative real-time RT-PCR. The expression level of Rab37 mRNA in the Si-control transfectant was set to 100 and the relative expression level in the Si-Rab37 transfectant is presented as mean ± SD (n = 3). ***p < 0.001. (B,C) Specificity of the Rab37 knockdown. Cell lysates (20 μg) from the G418-surviving cells transfected with Si-control or Si-Rab37 plasmid (C), together with HA-Rab37 or the knockdown-resistant HA-Rab37* plasmid (B) were subjected to western blot analyses with the antibodies indicated. Arrowheads indicate position of Rab27a and Rab27b (C). The full-length blotting images of (B,C) are presented in Supplementary Figs S4 and S5, respectively. The graphs represent the relative quantity of each Rab, with the quantity in the Si-control transfectant set to 100. Bars represent mean ± SD (n = 4). ***p < 0.001; NS, not significant. (D,E) Degranulation in the Rab37-knockdown cells. G418-surviving cells transfected with Si-Rab37 (squares; n = 4) or Si-control (triangles; n = 4) plasmid were sensitised with IgE and stimulated with (+Ag) or without (−Ag) antigen (D) or directly stimulated with (+A23187) or without (−A23187) A23187/TPA (E) for the indicated time periods. The degree of β-hexosaminidase release was measured and presented as in Fig. 1(C-F). *p < 0.05; **p < 0.01, compared to the stimulated Si-control transfectant. (F) Rescue experiment using HA-Rab37*. The degree of 10-minute β-hexosaminidase release from the G418-surviving cells transfected with Si-Rab37 or Si-control plasmid, together with HA-Rab37* or control (Vector) plasmid, was examined as in (D). After subtraction of the spontaneous release, the antigen-induced release from the Si-Rab37 transfectant with the control plasmid was set to 100, and the relative release from other transfectants compared to the Si-Rab37 transfectant with the control plasmid is presented as mean ± SD (n = 4). *p < 0.05; **p < 0.01.