FIGURE 2:

siRNA-based knockdown of PTPN23. Validation of the loss of nuclear SMN accumulation upon knockdown of PTPN23 (kd PTPN23) by forward transfection using three different siRNA oligonucleotides: Ambion silencer select s24775 (oligo1), s24777 (oligo2), and Stealth select HSS108537 (oligo3); siRNAs against vimentin were used as negative controls. Cells were transfected and incubated for 72 h. (A) Immunostaining of HeLa cells after siRNA transfection (oligo1) using α-SMN and α-coilin antibodies. Blue, DNA; green, SMN; red, coilin; scale bar, 20 μm. (B) Quantification of the loss of nuclear SMN accumulations observed in A. The relative numbers of cells bearing SMN accumulations (one or more) in Cajal bodies were calculated as mean values from three independent experiments (N = 200) and are represented in columns; error bars, SEM. (C) Western blot analysis of SMN and PTPN23 protein amounts after PTPN23 knockdown; HeLa total lysates were separated on a high-TEMED SDS gel (see Materials and Methods), and the SMN protein amount was analyzed by quantitative immunoblot using specific antibodies against SMN; numbers indicate relative SMN protein amounts; tubulin was used as a loading control. PTPN23 knockdown was analyzed using a 10% SDS–PAGE gel, flowed by Western blot using an α-PTPN23 antibody.