交互Nesprin-2-SMC和Nesprin-2 SMC2和SMC4。(一)降水SMC2和SMC4 GST-Nesprin-2-SMC HaCaT细胞溶解产物。沉淀是解决SDS-polyacrylamide凝胶(10%丙烯酰胺)和探测SMC2和SMC4特定抗体。SPN,下拉后上层清液;PD,下拉。的低分子量乐队SMC2下拉大概也是一个分解产物。(b)免疫沉淀反应的SMC2 HaCaT与Nesprin-2 mabk20 - 478和特定的细胞溶解产物Nesprin-2 SMC2特定抗体。GFP-specific单克隆抗体被用于控制。用于免疫沉淀反应的抗体在面板(IP)表示。探讨了这些墨迹与右边列出的抗体(WB)。 Immunoprecipitates were resolved on gradient gels (3–12% acrylamide) and 10% acrylamide gels as appropriate. The data are from one blot; however, the input was not directly adjacent to the SMC2 IP. (c) Interaction of CAP-H2 (condensin II) and CAP-H (condensin I) with Nesprin-2-SMC. Pulldowns were performed with HaCaT cell lysates and GST for control and GST-Nesprin-2-SMC as indicated. Unsynchronized cells were used for the experiments shown in (a)–(c). (d) Analysis of the Nesprin-2-SMC interaction with SMC2 during the cell cycle. HaCaT cells were synchronized with RO-3306 or other reagents as described in Materials and Methods in order to obtain the relevant cell cycle phases. Cell cycle phases were assessed by FACS analysis; the results are depicted in the accompanying diagram. Pulldown was carried out with GST-Nesprin-2-SMC bound to GST-Sepharose. GST was used for control. The blot was probed with SMC2 specific antibodies. (e) Localization of Nesprin-2 as detected with mAb K81-116-6 (green) during mitosis in HaCaT cells. DNA was stained with DAPI. Arrow points to filamentous staining across the chromosomes. (f) Nesprin-2 distribution in HaCaT cells during mitosis as detected with mAb K20-478 (green) and pAbK1 (red). DNA was detected with DAPI. Bar, 10 μm。(g) Nesprin-2出现在染色体。不同的Z-stacks(从上到下:0μ0.21米,μ0.42米μ0.84米,μ米)在COS7细胞后期沾马伯甘蓝型- 478。DNA与DAPI染色。酒吧,5μm。