Inhibition of Hsp90 influences AKT degradation and intracellular accumulation of rNadA.

A: Chang cells were incubated for 1, 4, 24 and 48 hours with either 17-AAG or FITC-GA at two different concentrations. At the end of each period cells were washed, trypsinized and RIPA-buffer total lysate prepared. Proteins from each extract were separated on NuSieve gel, blotted, and AKT or Actin (loading control) were detected using the respective primary antibodies. B: Chang cells were pre-treated with 0.01% saponin in PBS for 30 seconds at room temperature, washed three times with PBS and then handled as described in A. C: Chang cells were pre-treated for 1 hour with either vehicle (upper panels), 10 µM 17-AAG (middle panel) or 10 µM FITC-GA (lower panel), and then incubated with 200 µg/ml rNadA at 37°C for 1 (left panels) or 4 hours (right panels). Cells were then fixed, permeabilized and stained for rNadA. The drugs were present during the entire incubation period. IF intensity was calculated as mean ± s.e.m in two independent experiments, each assessing 10–15 cells and expressed as Arbitrary Units (A.U.). Scale bar 10 µm.

A：将Chang细胞（Chang cells）分别以两种不同浓度的17-AAG或FITC-GA处理，孵育时长分别为1、4、24及48小时。各孵育时段结束后，收集细胞并进行洗涤、胰酶消化，采用RIPA缓冲液（RIPA-buffer）制备全细胞裂解液。将各裂解液中的蛋白质在NuSieve凝胶（NuSieve gel）上进行电泳分离，随后转膜，使用对应一抗分别检测AKT（AKT）或肌动蛋白（Actin，上样对照）。
B：将Chang细胞在室温下用含0.01%皂苷（saponin）的磷酸盐缓冲液（PBS）预处理30秒，随后用PBS洗涤三次，后续实验操作同A组。
C：将Chang细胞分别用溶剂对照（上方面板）、10 μM 17-AAG（中间面板）或10 μM FITC-GA（下方面板）预处理1小时，随后加入200 μg/ml的重组NadA（rNadA），于37℃下分别孵育1小时（左侧面板）或4小时（右侧面板），整个孵育过程中均保留上述药物。孵育结束后对细胞进行固定、透化处理，并针对rNadA进行免疫荧光染色。免疫荧光强度以平均值±标准误（s.e.m.）表示，基于两次独立实验计算得到，每次实验统计10~15个细胞，结果以任意单位（Arbitrary Units，A.U.）呈现。标尺为10 μm。