Nesprin-2 Interacts with Condensin Component SMC2

Figure 4

Knockdown of Nesprin-2 using shRNA directed against C-terminal, N-terminal, and SMC domain sequences. (a) Western blots showing the efficiency of the shRNA treatment at the protein level. HaCaT cells were transfected with shRNAs targeting the various regions and for control (ctrl) with the corresponding scrambled shRNAs. Nesprin-2 at ~800 kDa was detected by mAb K20-478. Lamin B1 was used for loading control. (b) Immunofluorescence analysis of HaCaT cells treated with shRNAs targeting the C-terminus (Ne-2 C-term KD), the N-terminus (Ne-2 N-term KD), or the SMC domain (Ne-2 SMC KD). Cells were stained with antibodies directed against the N-terminus (mAb K20-478, green) and the C-terminus (pAbK1, red) of Nesprin-2. DAPI was used to visualize DNA. Arrowhead indicates cells with successful knockdown; asterisk indicates cells which still express Nesprin-2. Bar, 10 μm. (c) Immunolabelling of Ne-2 SMC KD cells with mAb K81-116-6. Nuclei were labeled with DAPI. Asterisk indicates a cell which still expresses Nesprin-2. Bar, 10 μM.

(a)

(b)

(c)