Fig. 7
From: 2-thio-6-azauridine inhibits Vpu mediated BST-2 degradation
Effect of 2-thio-6-azauridine on BST-2/Vpu/β-TrCP2 interaction. a Effect of 2-thio-6-azauridine on the interaction between Vpu and β-TrCP2. 293T cells were co-transfected with pEYFP-N1-Vpu, pRluc-C3-β-TrCP2 and pBST-2, which express EYFP tagged Vpu, RLuc tagged β-TrCP2 and HA tagged BST-2 respectively. 24 h post transfection, cells were treated with DMSO or 5 µM 2-thio-6-azauridine for 24 h. BRET ratios were measured as described in “Methods”. BRET ratios are normalized relative to those in control cells that were treated with DMSO (left panel). b 293T cells were co-transfected with pVphu, pcDNA-HOS-HA and pcDNA-BST-2-FLAG which express Vpu, HA tagged β-TrCP2 and Flag tagged BST-2 respectively. 24 h post transfection, cells were treated with DMSO and 5 µM 2-thio-6-azauridine for 24 h. Lysates were immunoprecipitated with 1 µg mouse anti-HA antibody followed by immunoblotting with rabbit anti-HA (top left panel) and Vpu antibody (bottom left panel). The pre-IP lysates represent 1 % of the IP input and were also immunoblotted for β-actin as a loading control (bottom right panel). c Effect of 2-thio-6-azauridine on the interaction between Vpu and BST-2. 293T cells were co-transfected with pRluc-C3-BST-2 and pEYFP-N1-Vpu, or pEYFP-N1-Vpu (S52/56N), which express Rluc tagged BST-2, EYFP tagged Vpu and EYFP tagged Vpu (S52/56N) respectively. 24 h post transfection, cells were treated with DMSO and 5 µM 2-thio-6-azauridine for 24 h. BRET ratios were measured as described in “Methods”. Values presented here are the normalized BRET ratio relative to that treated with DMSO. The bar graphs represent the means of results of experiments performed at least three times, and the error bars represent standard deviations. d 293T cells were co-transfected with pVphu, pBST-2 (1:1). 24 h post transfection, cells were treated with DMSO and 5 µM 2-thio-6-azauridine for 24 h. Lysates were immunoprecipitated with 1 µg mouse anti-HA antibody followed by immunoblotting with BST-2 (top left panel) and Vpu antibodies (bottom left panel). The pre-IP lysates represent 1 % of the IP input and were also immunoblotted for β-actin as a loading control (bottom right panel)