Fig. 8

FKBP9 is degraded during Tg-induced ER stress. a SF-539 and T98G cells were pretreated with vehicle, GSK260414 (1 μM), ISRIB (100 nM) or 4μ8C (50 μM), and then exposed to vehicle, 0.5 μM Tg or 2.5 μM Tm for 6 h. IB analysis for FKBP9 and protein levels in UPR. b SF-539 and T98G cells were treated with vehicle, 0.5 μM Tg or 2.5 μM Tm for 6 h, and MG132 (1 μM), Baf A1 (10 nM), CQ (5 μM) were added to the cells 1 h in advance. Expression of FKBP9 was tested by IB using GAPDH as a loading control. c HEK-293 T cells were transfected with V5-tagged FKBP9, HA-tagged Ub, and then they were treated with Tg for 6 h. Cells were pretreated with MG132 the same as in (b). Immunoprecipitation (IP) was performed with anti-V5 antibody, IB with the indicated antibodies. d HEK-293 T cells were tranfected with HA-tagged Ub, V5-tagged FKBP9 wide-type and mutants, and then they were treated with Tg for 6 h. Similar analysis were performed as in (c) for FKBP9 ubiquitination. e T98G cells were transfected with V5-tagged FKBP9 wide type and mutants, and then treated with Tg. The ratios of V5 expression to their corresponding GAPDH were represented. f IB analysis for Caspase-12 and CHOP expression in SF-539-FKBP9-WT and SF-539-FKBP9-K265R cells exposed with Tg for 12 h. The ratios of Cleaved-Caspase-12, CHOP and V5 expression to their corresponding GAPDH were represented. All experiments in this figure were performed three times with comparable results