Fig. 6

AQP5 promotes ULK1 ubiquitination via TRIM21. a-d AQP5 was overexpressed or knocked down in 293 T cells; and the cells were transfected with ULK1-HA for 48 h. The cell lysates were subjected to IP with anti-HA agarose and immunoblotted with the indicated antibodies. (e and f) AQP5 was overexpressed in AGS cells, and the cells were co-transfected with UBB/UBC siRNA, K63-Ub-HA or K63-R-HA. IP assay was performed with anti-HA agarose, followed by immunoblotting with the indicated antibodies (e); Cell lysates were analyzed by WB with the indicated antibodies (h). (g and h) AQP5 was overexpressed in AGS cells transfected with ULK1-HA and TRIM21 siRNA, and the cells were analyzed by IP assays with anti-HA agarose and immunoblotted with the indicated antibodies (g) or (h) analyzed by WB with the indicated antibodies. (i and j) AQP5-overexpressing (i) and AQP5-knockdown (j) AGS cells were co-transfected with ULK1-HA and TRIM21-Myc. Cell lysates were subjected to IP with anti-HA agarose and immunoblotted with the indicated antibodies. (k) AQP5-overexpressing AGS cells were co-transfected with ULK1-HA and TRIM21-Myc. Confocal microscopy was used to analyze the interaction between TRIM21 (red) and ULK1 (green)