Fig. 3: FKBP39 associates with Nprl3 and promotes Nprl3 degradation.

A S2 cells were co-transfected with HA-tagged Nprl3 and V5-tagged FKBP39 or lacZ (control) plasmids. Cells were lysed and immunoprecipitated using beads coated with anti-HA antibody. Cell lysates (inputs) and immunoprecipitates (IP) were detected by western blot. B S2 cells were co-transfected with HA-tagged Nprl3 and V5-tagged FKBP39 or lacZ (control) plasmids. Cells were cultured in Schneider’s medium plus 10% FBS (AA+) or amino-acid-free Schneider’s medium (AA−) for 2 h. The cells were lysed and immunoprecipitated using beads coated with an anti-HA antibody. Cell lysates (inputs) and immunoprecipitates (IP) were detected by western blot. C S2 cells were transfected with GFP (control) or fkbp39 dsRNA. Cells were treated with amino acid starvation for 2 h. The levels of Nprl3 were detected by western blot. D S2 cells were co-transfected with the GFP (control) or fkbp39 dsRNA, constructs expressed HA-tagged Nprl3 and V5 tagged lacZ. The levels of HA-Nprl3 and V5-lacZ were detected by western blot. V5-lacZ was used as a loading control for western blot. E S2 cells were co-transfected with the GFP (control) or fkbp39 dsRNA and construct expressed V5-tagged Nprl3. Cells were treated with amino acid starvation for the indicated hours. The levels of V5-Nprl3 were detected by western blot. F S2 cells were co-transfected with the GFP (control) or fkbp39 dsRNA and construct expressed V5-tagged Nprl3. The cells were treated with CHX for the indicated time and the V5-Nprl3 levels were measured by western blot. The remaining protein levels at each time point were calculated, taken the expression level of V5-Nprl3 at 0 h as 1. Error bars represent standard error values from three independent experiments. *P < 0.05. α-Tubulin was used as a loading control for western blot. Similar western blot results were observed in more than three independent experiments for each group.