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. 2022 May 9;18(5):e1010506. doi: 10.1371/journal.ppat.1010506

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© 2022 Jia et al

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Fig 4 — (A) Interactions among Pns11, ATG5 and ATG12 were detected by yeast two-hybrid assay. Transformants were plated on either DDO or QDO+X-α-gal culture medium. Experiments were labeled as follows: Pns11/ATG5, pGBKT7-Pns11/pGADT7-ATG5; ATG5/ATG12, pGBKT7-ATG5/pGADT7-ATG12; Pns11/ATG12, pGBKT7-Pns11/pGADT7-ATG12; Positive control, pGBKT7-53/pGADT7-T; Negative control, pGBKT7-Lam/pGADT7-T. (B) The interactions among Pns11, ATG5 and ATG12 were detected by GST pull-down assay. GST-ATG5 or GST-Pns11 was incubated with glutathione-Sepharose beads. His-Pns11 or His-ATG12 was then added to the beads. Next, western blot assay was performed to detect His-Pns11 bound to GST-ATG5, His-ATG12 bound to GST-ATG5, and His-ATG12 bound to GST-Pns11. (C-G) Interactions among Pns11, ATG5 and ATG12 were detected in Sf9 cells. (C) At 48 hpi, Sf9 cells singly expressed with ATG5-His, ATG12-Flag or Pns11 were fixed and immunolabeled with His-rhodamine (red), Flag-rhodamine (red) or Pns11-rhodamine (red), respectively. (D) At 48 hpi, Sf9 cells co-expressed with ATG5-His and Pns11 were fixed and immunolabeled with His-FITC (green) and Pns11-rhodamine (red). (E) At 48 hpi, Sf9 cells co-expressed with ATG5-His and ATG12-Flag were fixed and immunolabeled with His-rhodamine (red) and Flag-FITC (green). (F) At 48 hpi, Sf9 cells co-expressed with Pns11 and ATG12-Flag were fixed and immunolabeled with Pns11-rhodamine (red) and Flag-FITC (green). (G) At 48 hpi, Sf9 cells triply-expressed with ATG5-His, ATG12-Flag and Pns11 were fixed and immunolabeled with Pns11-FITC (green), His-rhodamine (red), and Flag-Alexa Fluor 647 (blue). Bars, 5 μm.