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. 2014 Mar 6;15(4):392–401. doi: 10.1002/embr.201337995

Figure 2.

Figure 2

TBC1D5 is required for ATG9 trafficking.
  1. Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5. Cells were treated with DMSO or KU0063794 (6 h), lysed in RIPA buffer (1% SDS) and lysates were subjected to SDS–PAGE.
  2. U2OS cells stably expressing mCherry-ATG9 were depleted of VPS29 and TBC1D5 using shRNAs, treated with DMSO or KU0063794 (6 h), fixed with 2% PFA, and immunostained with anti-LAMP1 antibody.
  3. Quantification of ATG9 vesicular structures in cells from (B) under non-stimulated conditions.
  4. Quantification of ATG9 and LAMP1 co-localization in cells presented in (B).
  5. Quantification of ATG9 and LAMP1 co-localization in cells stably depleted for TBC1D5 (shRNA#1) or VPS29 (shRNA#2), transiently transfected with myc-TBC1D5 plasmid resistant to TBC1D5 shRNA#1. Cells were treated with KU0063794 (6 h), 20 h post-transfection, fixed, and immunostained with anti-LAMP1 and anti-myc antibodies.
  6. Control shRNA and U2OS cell lysates depleted for TBC1D5 (shRNA#1), transfected with empty plasmid or with shRNA-resistant myc-TBC1D5.
  7. Immunofluorescence of TBC1D5 (shRNA#1) cells transiently transfected with shRNA-resistant myc-TBC1D5, treated with KU0063794 (6 h). 20 h post-transfection cells were treated, subsequently fixed, and immunostained with anti-myc and anti-LAMP1 antibodies.

Data information: Bar graphs (C, E), mean ± s.d. n = 3, unpaired two-tailed t-test, P-values: ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Bar graph (D), mean ± s.d. n = 3, one-way ANOVA with Bonferroni′s multiple comparison test, ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.