PMC

HeLa cells were transfected to express KChIP1–EYFP or KChIP1EF2–4–EYFP as indicated, fixed and immunostained with mouse monoclonal antibodies specific for membrin (A), GOS28 (B), VAMP7 (C and E) or Vti1a (D and F). The images show the localization of the KChIP1 and the indicated SNARE proteins and the colour overlays show KChIP1–EYFP in green and anti-SNARE staining in red with co-localization appearing in yellow. The scale bars represent 10 μm in the main Figures and 2 μm in the enlarged inserts.

HeLa cells were co-transfected to express both KChIP1–EYFP and Kv4.2 and immunostained with anti-Kv4.2 so that localization of KChIP1 (A) and Kv4.2 (B) could be visualized. Alternatively, the cells were additionally co-transfected to express the Rab1 GAP TBC1D20 (D–F). The colour overlays (C and F) show co-expressed proteins in green (KChIP1–EYFP) and red (Kv4.2), with co-localization appearing in yellow. The scale bars represent 10 μm. Normalized traffic of Kv4.2 in the absence (cont) or presence (GAP) of TBC1D20 was quantified and the data shown as means±S.E.M. from 20 cells for each condition (G).

HeLa cells were transfected to express KChIP1–EYFP, fixed and immunostained with anti-VAMP7 in the absence (A) or presence (B) of VAMP7 siRNA so that localization of KChIP1 and VAMP7 could be visualized. The colour overlays show co-expressed proteins in green (KChIP1–EYFP) and red (VAMP7) with co-localization appearing in yellow. The scale bars represent 10 μm. (C) For quantitative PCR, mRNA was prepared from HeLa cells transfected in the presence of silent or VAMP7 siRNA and relative mRNA levels determined using real-time PCR. RNAi, RNA interference.

HeLa cells were transfected to express KChIP1–EYFP, fixed and immunostained with mouse monoclonal antibodies specific for syntaxin 7 (A–C) or syntaxin 8 (D–F). The images show that KChIP1 punctae are distinct from those stained by anti-syntaxin 7 or 8. In cells immunostained with anti-VAMP 7 and anti-LAMP1 (G–I), there was little overlap of the stained punctae. The colour overlays show KChIP1–EYFP or anti-VAMP7 in green and anti-syntaxin 7, 8 or anti-LAMP1 in red, with co-localization appearing in yellow. The scale bars represent 10 μm in the main Figures and 2 μm in the enlarged inserts.

(A) HeLa cells were co-transfected to express both KChIP1–EYFP and Kv4.2 and immunostained with anti-Kv4.2 so that localization of KChIP1 and Kv4.2 could be visualized. Control cells are shown as well as cells co-transfected with VAMP7 or Vtila siRNA as indicated. The colour overlays show co-expressed proteins in green (KChIP1–EYFP) and red (Kv4.2) with co-localization appearing in yellow. The scale bars represent 10 μm. Normalized traffic of Kv4.2 in control cells and cells co-transfected with Vti1a (B) or VAMP7 (C) siRNAs was quantified and the results shown as means±S.E.M. from 20 (Vti1A) or 25 (VAMP7) cells for each condition. cont, control; RNAi, RNA interference.

HeLa cells were transfected to express VSVG–GFP and imaged. Control cells are shown as well as cells co-transfected with silent, VAMP7 or Vtila siRNA as indicated (A). Normalized traffic of VSVG in control cells (cont) and cells co-transfected with silent, Vti1a or VAMP7 siRNAs was quantified and the results are shown from 20 cells for each condition (B). HeLa cells were transfected to express KChIP2 and Kv4.2 and immunostained with anti-Kv4.2 to allow imaging of Kv4.2 localization. Control cells are shown as well as cells co-transfected with silent, VAMP7 or Vtila siRNA as indicated (C). Normalized traffic of Kv4.2 in control cells and cells co-transfected with silent, Vti1a or VAMP7 siRNAs was quantified and the results shown as means±S.E.M. from 30 cells for each condition (D). The scale bars represent 10 μm. RNAi, RNA interference.

(A) Neuro2A cells were transfected to co-express Kv4.2 and EYFP–Golgi. (B and C) Neuro2a cells were transfected to express KChIP1–EYFP, fixed and immunostained with anti-Vti1a or anti-VAMP7 as indicated. (D) Neuro2a cells were co-transfected to express both KChIP1-EYFP and Kv4.2 and immunostained with anti-Kv4.2 so that localization of KChIP1 and Kv4.2 could be visualized. Control cells are shown as well as cells co-transfected with silent, VAMP7 or Vtila siRNA as indicated. (E) Normalized traffic of Kv4.2 in control cells (cont) and cells co-transfected with siRNAs was quantified and the results shown as means±S.E.M. from 20 cells for each condition. The scale bars represent 10 μm. RNAi, RNA interference.

HeLa cells were transfected to co-express Kv4.2 and EYFP–Golgi (A–C) or ARF1–EYFP (D–F) or with wild-type KChIP1-EYFP (G), with the single EF hand mutant KChIP1EF3-EYFP (H) or with the triple EF-hand mutant KCHIP1EF2–4-EYFP (I) and Kv4.2 localization was determined by using anti-Kv4.2 immunostaining. HeLa cells were transfected to express VSVG–GFP alone (J), or with wild-type KChIP1 (K) or the triple EF-hand mutant KCHIP1EF2–4-HcRed (L) and VSVG–GFP localization was determined by confocal imaging. The scale bars represent 10 μm. Normalized traffic of Kv4.2 (M) or VSVG–GFP (N) in the presence of various KChIPs was quantified and the results shown as means±S.E.M. from 20 cells for each condition. w/t, wild-type.