Tid1 mediates autophagy independently of its interaction with Hsp70.
A, vector, Tid1S-FLAG, HSP70-HA, or Tax-HA was transiently transfected into HT1080 cells, and total protein lysates were collected 24 h post-transfection for detecting LC3, Tid1S-FLAG, HSP70-HA, or Tax-HA using immunoblots. B, Tid1S-FLAG or Tid1SΔJ-FLAG was transiently co-transfected with HSP70-GST. 24 h post-transfection, total protein extracts were prepared for GST pulldown assay. C, anti-LC3 blot in HT1080 cells transfected with vector, Tid1S-FLAG, Tid1SΔJ-FLAG, or Tax-HA. D, GST pulldown assay to evaluate the interaction of HSP70 with Tid1L-FLAG or with Tid1LΔJ-FLAG. E, the average numbers of GFP-LC3 puncta per cell in HT1080 cells transfected with Tid1S-FLAG, Tid1SΔJ-FLAG, or Tax-HA. **, p < 0.01. F, HOS/FLAG-LC3 cells were transduced with control lentivirus or the lentivirus expressing Tid1L-FLAG, Tid1S-FLAG, or Tax-HA. 4 days post-transduction, immunoblot analysis was performed to detect proteins indicated in the figure.