Figure 4. AP-1 and KIF13A function... | Rockefeller University Press

Figure 4.

$Figure 4. AP-1 and KIF13A function together on the peripheral localization of recycling endosomes. (A) MNT-1 cells were fixed in cold methanol, and endogenous KIF13A localization (a and d) was assessed relative to AP-1 (b) or Tf internalized for 45 min (f). Single labeling is shown in columns 1 and 2, merged images in column 3, and magnified insets (of boxed areas) in column 4. KIF13A-labeled tubular and punctate structures partially overlap with γ-adaptin (d, arrows) and internalized Tf (h, arrows). (B) Western blot analysis of lysates of cells treated with control (Ctrl), AP-1 (µ1a), or KIF13A siRNAs using anti-KIF13A or anti-µ1A antibodies or anti–β-tubulin as a loading control. (C)Anti-KIF13A or γ-adaptin immunoprecipitates from MNT-1 cell lysates were fractionated by SDS-PAGE and immunoblotted using KIF13A antibodies. Anti-KIF13A immunoprecipitates contain two bands corresponding to the monomeric and dimeric forms. Anti–γ-adaptin immunoprecipitates contain only the monomeric form. (D) MNT-1 cells were treated with control, µ1A, β3A, or KIF13A siRNAs and analyzed by IFM after internalization of Tf-A488. The intensity of fluorescence in the peri-Golgi area was evaluated as a compaction factor. Data were normalized to the control and presented as mean ± SD. (E) After internalization of Tf-A488 (a and e), control (a–d) and KIF13A (e–h) siRNA–treated cells were fixed and double labeled for γ-adaptin (b and f) and Rab11 (c and g). Depletion of KIF13A results in peri-Golgi clustering of Tf-A488– and Rab11-positive endosomes (e, g, and h, arrows). Merged images and magnified insets (of boxed areas) are shown in column 4. (F) IFM analysis of control (a–c) and KIF13A (d–f) siRNA–treated cells that were double labeled for TGN46 (a and d) and γ-adaptin (b and e). (G) The distribution of γ-adaptin labeling in the vicinity of the Golgi was measured in cells treated with control or KIF13A siRNAs. (B and C) Molecular masses are given in kilodaltons. Bars, 10 µm.$

AP-1 and KIF13A function together on the peripheral localization of recycling endosomes. (A) MNT-1 cells were fixed in cold methanol, and endogenous KIF13A localization (a and d) was assessed relative to AP-1 (b) or Tf internalized for 45 min (f). Single labeling is shown in columns 1 and 2, merged images in column 3, and magnified insets (of boxed areas) in column 4. KIF13A-labeled tubular and punctate structures partially overlap with γ-adaptin (d, arrows) and internalized Tf (h, arrows). (B) Western blot analysis of lysates of cells treated with control (Ctrl), AP-1 (µ1a), or KIF13A siRNAs using anti-KIF13A or anti-µ1A antibodies or anti–β-tubulin as a loading control. (C)Anti-KIF13A or γ-adaptin immunoprecipitates from MNT-1 cell lysates were fractionated by SDS-PAGE and immunoblotted using KIF13A antibodies. Anti-KIF13A immunoprecipitates contain two bands corresponding to the monomeric and dimeric forms. Anti–γ-adaptin immunoprecipitates contain only the monomeric form. (D) MNT-1 cells were treated with control, µ1A, β3A, or KIF13A siRNAs and analyzed by IFM after internalization of Tf-A488. The intensity of fluorescence in the peri-Golgi area was evaluated as a compaction factor. Data were normalized to the control and presented as mean ± SD. (E) After internalization of Tf-A488 (a and e), control (a–d) and KIF13A (e–h) siRNA–treated cells were fixed and double labeled for γ-adaptin (b and f) and Rab11 (c and g). Depletion of KIF13A results in peri-Golgi clustering of Tf-A488– and Rab11-positive endosomes (e, g, and h, arrows). Merged images and magnified insets (of boxed areas) are shown in column 4. (F) IFM analysis of control (a–c) and KIF13A (d–f) siRNA–treated cells that were double labeled for TGN46 (a and d) and γ-adaptin (b and e). (G) The distribution of γ-adaptin labeling in the vicinity of the Golgi was measured in cells treated with control or KIF13A siRNAs. (B and C) Molecular masses are given in kilodaltons. Bars, 10 µm.