Effect of C3bot1E174Q-C2I on primary cultured macrophages.

Monocytes from human blood were differentiated for 7 days. The resulting macrophages were treated for 6 h at 37°C with C3bot1E174Q-C2I (4 µg/mL) or for control with C2I+C2IIa (200+400 ng/mL) or left untreated. Cells were lysed and lysates incubated for 30 min at 37°C with C2I (300 ng) and biotin-labelled NAD+ (10 µM) to ADP-ribosylate unmodified actin. Comparable amounts of lysate protein were confirmed by SDS-PAGE and Coomassie staining (not shown). The biotinylated (i.e. ADP-ribosylated) actin was detected by Western blotting with streptavidin-peroxidase. B. Intensity of the bands showing ADP-ribosylated actin was determined by densitometry and is given as percentage of actin from untreated control cells (mean±S.D.; n = 3; * = p≤0.5, ** = p≤0.05). B. Comparable amounts of total protein in the lanes were confirmed by Ponceau S staining and anti-Hsp90 Western blotting. B. Morphology of the cells described in A after 6 h. C. HeLa cells were incubated with C3bot1E174Q-C2I (4 µg/mL)+C2IIa (8 µg/mL) or with C3bot1E174Q-C2I alone (4 µg/mL). For control cells were left untreated or were incubated with C2I alone (4 µg/mL). After 6 h of incubation at 37°C all cells were washed, incubated with an antibody against C2I for 5 min at 4°C to remove non-internalized C2I and C2I fusions, washed again and pictures were taken.

从人血液中分离的单核细胞经7天诱导分化，所得巨噬细胞于37℃下分别用C3bot1E174Q-C2I（4 µg/mL）处理6小时，或采用C2I+C2IIa（200 ng/mL + 400 ng/mL）作为对照处理，同时设置未处理组。收集细胞并裂解，将裂解液于37℃下与C2I（300 ng）及生物素标记的烟酰胺腺嘌呤二核苷酸（NAD+，10 µM）共同孵育30分钟，以对未修饰的肌动蛋白进行ADP核糖基化修饰。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及考马斯亮蓝染色确认各组裂解液蛋白上样量一致（数据未展示）。采用链霉亲和素-过氧化物酶免疫印迹（Western blotting）检测经生物素标记（即ADP核糖基化修饰）的肌动蛋白。
B. 通过光密度扫描法测定显示ADP核糖基化肌动蛋白的条带灰度值，以未处理对照组细胞的肌动蛋白含量为基准计算相对百分比（结果以平均值±标准差表示，n=3；*表示p≤0.5，**表示p≤0.05）。
B. 采用丽春红S染色及抗热休克蛋白90（Hsp90）免疫印迹验证各组泳道总蛋白上样量一致。
B. 展示A部分所述细胞经6小时处理后的形态学特征。
C. 将HeLa细胞于37℃下分别以C3bot1E174Q-C2I（4 µg/mL）+C2IIa（8 µg/mL）、单独的C3bot1E174Q-C2I（4 µg/mL）进行处理，对照组设置未处理组及单独C2I（4 µg/mL）处理组。于37℃孵育6小时后，洗涤所有细胞，于4℃下用抗C2I抗体孵育5分钟以去除未内化的C2I及C2I融合蛋白，再次洗涤后拍摄显微图像。