Fig 1: Schematic representation of the role of TIM-4 N-glycosylation in NSCLC metastasis. TIM-4 is N-glycosylated at Asn291 and TIM-4 promotes NSCLC metastasis by modifying N-glycans at Asn291. When the N-glycosylation of TIM-4 is removed, the stability of TIM-4 protein is decreased and TIM-4 is more susceptible to degradation by ER-localized ubiquitin ligase-mediated ERAD. Thus, the function of TIM-4-mediated metastasis of NSCLC cells was suppressed.
Fig 2: N-glycosylated TIM-4 promotes metastasis of NSCLC cells. Wound healing assay was performed to evaluate the migration abilities of TIM-4-transfected and N291Q-transfected A549 cells (A) and H1299 cells (B). Transwell assay was performed to evaluate the migration and invasive abilities of TIM-4-transfected and N291Q-transfected A549 cells (C) and H1299 cells (D). (Scale bar:100µm) All error bars were shown as mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001, by Student’s t-test.
Fig 3: Effects of N-glycosylation on location and degradation pathway of TIM-4. (A) Confocal immunofluorescence microscopy was used to detect location of TIM-4 in TIM-4-transfected and N291Q-transfected A549 cells. (B) Confocal immunofluorescence microscopy was used to detect colocalization between TIM-4 and ER in TIM-4-transfected and N291Q-transfected A549 cells. (Scale bar:10µm). (C) Western Blot was used to detect the degradation pathway of TIM-4.
Fig 4: N-glycosylation increases stability of TIM-4. (A) Western Blot was used to detect the stability of TIM-4 before and after the removal of N-glycosylation. (B) Western Blot was used to detect the degradation pathway of TIM-4 before and after the removal of N-glycosylation. (C) Ubi-HA was transfected into A549-LV-TIM-4-Flag cells, and TM or MG132 was used to treat cells. TIM-4 was precipitated by anti-Flag, and Western Blot was used to detect the ubiquitination of TIM-4 before and after the removal of N-glycosylation. (D) A549 cells were transfected with pcDNA3, TIM-4-HA or N291Q-HA respectively and treated with MG132. TIM-4 was precipitated by anti-HA, and Western Blot was performed to detect the ubiquitination of TIM-4. **p < 0.01, ***p < 0.001, by Student’s t-test.
Fig 5: N-glycosylated TIM-4 promotes metastasis of NSCLC cells in vivo. (A) Experimental flow chart and design. (B) Western Blot was used to detect the expression of TIM-4 protein. (C) Photographs of nude mice for in vivo imaging. (D) Statistics of nude mice for in vivo imaging. *p < 0.05, by Student’s t-test.
Supplier Page from MilliporeSigma for Anti-TIMD4 antibody produced in rabbit