Extended Data Fig. 10: KIF5B is required for ERGIC compartmentalization.
From: A dual role of ERGIC-localized Rabs in TMED10-mediated unconventional protein secretion
a, The HEK293T cells stably expressing mIL-1β-HA were transfected with indicated shRNA plasmids. 72h post transfection, the cells were harvested for knockdown efficiency test by qPCR. b, Immunofluorescence and SIM analysis of endogenous KIF5B, TMED10-V5 and BFP/BFP-Rab1A/1B in HeLa cells using anti-KIF5B and anti-V5 antibody. Scale bar, 5 μm in overview and 2 μm in the zoomed in. Diagram showing the percentage of KIF5B with TMED10 (mean ± SD). P values are indicated (two-tailed t test, n=50 cells, ns, no significant difference). c, Immunofluorescence and SIM analysis of endogenous KIF5B, TMED10-V5 and BFP/BFP-Rab2A in HeLa cells transfected with control shRNA (sh Ctr) or shRNA plasmids against Rab1A/1B/2B (sh Rab1A/1B/2B) using anti-KIF5B and anti-V5 antibody. Scale bar, 5 μm in overview and 2 μm in the zoomed in. Diagram showing the percentage of KIF5B with TMED10 (mean ± SD). P values are indicated (two-tailed t test, n=50 cells, ns, no significant difference). d–f, ssGFP (d)/IL-6 (e)/IL-10 (f) secretion in HEK293T cells co-transfected with GFP/KIF5B-GFP plasmids. g–i, ssGFP (g)/IL-6 (h)/IL-10 (i) secretion in HEK293T cells co-transfected with control shRNA (Ctr) or shRNA plasmids against KIF5B (KIF5B KD). j, The HEK293T cells of WT, KIF5B KD and Rab1B KD were transfected with GFP(1–10)-TMED10 and IL-1β-GFP11. FACS analysis was performed to determine the complemented GFP signal with cells that did not have IL-1β-GFP11 expression as a negative control. Diagram showing the quantification of GFP signal (mean ± SD). P values are indicated (two-tailed t test, n=3 experiments, ns, no significant difference). Immunoblot showing the expression of the indicated proteins. k, CoIP analysis using HEK293T cells with GFP/GFP–Rab2A and endogenous KIF3B. l, CoIP analysis using HEK293T cells with TMED10-V5 and endogenous KIF5B with or without BFP-Rab2A overexpression. *, Non-specific band. d–i,k,l, The data are representative of three independent experiments. Exact P values are presented in Source numerical data. Source numerical data and unprocessed blots are available in Source data.
