743 amino acids  (Addgene inc)

Journal: Molecular Biology of the Cell

Article Title: The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization

doi: 10.1091/mbc.E14-06-1151

Figure Lengend Snippet: 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.

Article Snippet: We loaded 3 mg of total protein from HeLa lysate onto 50 μl of PTPN23 peptide antibody (amino acids 710–743) covalently coupled to protein A–Sepharose beads (Life Technologies) and incubated for 2 h by rotation at 4°C.

Techniques: Transfection, Incubation, Immunostaining, Western Blot, SDS-Gel, SDS Page

Journal: Molecular Biology of the Cell

Article Title: The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization

doi: 10.1091/mbc.E14-06-1151

Figure Lengend Snippet: Kinetics of PTPN23 depletion upon siRNA. Kinetics of PTPN23 protein depletion using different siRNAs as explained for ; siRNAs against vimentin were used as negative controls. Cells were transfected, and samples were taken every 24 h for a period of 3 d. (A) Western blot analysis of SMN and PTPN23 protein amounts after siRNA-mediated PTPN23 knockdown; the PTPN23 and SMN protein amounts were analyzed by quantitative immunoblot using specific antibodies against SMN and PTPN23; numbers indicate time points after siRNA transfection. Tubulin was used as a loading control. (B) Graphs represent amounts of PTPN23 or SMN over a 3-d period evaluated as determined by quantitative Western blot. (C) Increase in number of cells losing SMN accumulations in CBs.

Article Snippet: We loaded 3 mg of total protein from HeLa lysate onto 50 μl of PTPN23 peptide antibody (amino acids 710–743) covalently coupled to protein A–Sepharose beads (Life Technologies) and incubated for 2 h by rotation at 4°C.

Techniques: Transfection, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization

doi: 10.1091/mbc.E14-06-1151

Figure Lengend Snippet: Interaction of SMN and PTPN23. Immunoprecipitation was performed in HeLa total cell lysates using an α-PTPN23 antibody (A, B) covalently coupled to protein A or G–Sepharose; an unspecific rabbit IgG coupled to protein A–Sepharose was used as a control. (A) SDS–PAGE and colloidal Coomassie stain of α-PTPN23 and α-IgG immunoprecipitation; the most prominent band was identified as PTPN23 by mass spectrometry. (B) Immunoblot analysis of immunoprecipitations using α-PTPN23 antibodies or an unspecific IgG as a controls. SMN, unrip, PTPN23, and tubulin were detected. sup, supernatant. The asterisk indicates an unspecific band.

Article Snippet: We loaded 3 mg of total protein from HeLa lysate onto 50 μl of PTPN23 peptide antibody (amino acids 710–743) covalently coupled to protein A–Sepharose beads (Life Technologies) and incubated for 2 h by rotation at 4°C.

Techniques: Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization

doi: 10.1091/mbc.E14-06-1151

Figure Lengend Snippet: Subcellular localization of exogenous and endogenous PTPN23. (A) HA-tagged PTPN23 was expressed in HeLa cells for 6, 8, and 16 h and detected by Western blot using specific antibodies against the HA tag (left) or PTPN23 (right). (B) Localization analysis of HA-PTPN23 by indirect immunofluorescence in cells expressing the protein for 8 h (blue, DAPI; red, HA; scale bar, 20 μm). (C) Localization analysis of endogenous PTPN23 by indirect immunofluorescence using an α-PTPN23 antibody; blue, DAPI; red, PTPN23; scale bar, 20 μm. (D) Localization analysis of HA-PTPN23 (8-h expression) before and after addition of the nuclear protein export inhibitor LMB. LMB was applied at 45 min; indirect immunofluorescence was performed using an α-HA antibody; blue, DAPI; red, HA; scale bar, 20 μm. White lines indicate fluorescence intensity measurements; plots show the respective intensity analysis after ImageJ-based quantification. Measured sections in the cytoplasm, within the nucleoplasm, and in the nucleolus are indicated within the plots in different colors.

Article Snippet: We loaded 3 mg of total protein from HeLa lysate onto 50 μl of PTPN23 peptide antibody (amino acids 710–743) covalently coupled to protein A–Sepharose beads (Life Technologies) and incubated for 2 h by rotation at 4°C.

Techniques: Western Blot, Immunofluorescence, Expressing, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization

doi: 10.1091/mbc.E14-06-1151

Figure Lengend Snippet: Assembly and phosphorylation state of SMN after PTPN23 knockdown. (A) Association of SMN with interaction partners in the complex in control cell lysates or lysates after PTPN23 knockdown. SMN was immunoprecipitated from total lysates using specific SMN-antibodies, and immunoprecipitates were purified and probed for the presence of SMN, Gemin3, Gemin 4, and unrip as indicated. (B) Two-dimensional gel electrophoresis and immunoblot analysis of SMN after PTPN23 knockdown (kd). Cells were transfected with siRNAs against the indicated phosphatases; vimentin was used as a negative control. At 72 h after transfection, cells were resuspended in isoelectric focusing buffer and lysed; proteins corresponding to 10-mg cell pellet were used for isoelectric focusing, subsequent SDS–PAGE, and Western blot analysis. The phosphorylation state of the SMN protein was analyzed using an antibody specific to SMN in controls or after PTPN23 knockdown. Red arrows in bottom (PTPN23 knockdown) indicate disappearing phosphorylated SMN species.

Article Snippet: We loaded 3 mg of total protein from HeLa lysate onto 50 μl of PTPN23 peptide antibody (amino acids 710–743) covalently coupled to protein A–Sepharose beads (Life Technologies) and incubated for 2 h by rotation at 4°C.

Techniques: Immunoprecipitation, Purification, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Transfection, Negative Control, SDS Page