Journal: Journal of Biological Chemistry

Article Title: Phosphatidylinositol Phosphate Kinase Type Iγ Directly Associates with and Regulates Shp-1 Tyrosine Phosphatase

doi: 10.1074/jbc.m500576200

Figure Lengend Snippet: FIG. 1. PIPKI661 directly interacts with Shp-1. A, reciprocal GST pull-down of PIPKI661 and Shp-1. T7-tagged PIPKI661 was incubated with GST-Shp-1 or GST alone, and the complexes were pelleted with glutathione-Sepharose beads. Bound PIPKI661 was an- alyzed by Western blot using an antibody specific for the T7 epitope tag. T7-tagged Shp-1 was incubated with GST-PIPKI661, and the pelleted complex was analyzed in the same manner. The GST-tagged proteins were blotted with an antibody specific for GST. B, HEK293 cells were lysed, and Shp-1 was immunoprecipitated with a monoclonal antibody specific for Shp-1. The immunoprecipitates were resolved via SDS- PAGE and analyzed by Western blot as indicated. Normal mouse IgG was used as a control. C, HEK293 cells were co-transfected with 2 g of PIPKI661 and 2 g of Shp-1 via calcium phosphate. After 48 h, cells were subjected to immunoprecipitation assays using polyclonal antibod- ies specific for the C terminus of PIPKI661 or a monoclonal anti-Shp-1 antibody. The immunoprecipitates were resolved and probed with the antibodies indicated. Normal mouse and rabbit IgGs were used as controls.

Article Snippet: Horseradish peroxidase-conjugated anti-GST antibody was purchased from Amersham Biosciences, and horseradish peroxidase-conjugated anti-T7 an- tibody was obtained from Novagen.

Techniques: Incubation, Western Blot, Immunoprecipitation, SDS Page, Control, Transfection

Journal: Journal of Biological Chemistry

Article Title: Phosphatidylinositol Phosphate Kinase Type Iγ Directly Associates with and Regulates Shp-1 Tyrosine Phosphatase

doi: 10.1074/jbc.m500576200

Figure Lengend Snippet: FIG. 2. The phosphatase domain of Shp-1 binds to the C termi- nus of PIPKI. A, HEK293 cells were co-transfected with 2 g of either Shp-1 or Shp-2 and 2 g of the indicated PIPKI constructs. After 48 h, Shp-1 and Shp-2 were immunoprecipitated with monoclonal antibodies specific for the respective phosphatase. The immunoprecipitates were then analyzed by Western blot. The images blotting for immunoprecipi- tated PIPKI were taken from the same blot and exposure time. B, HEK293 cells were co-transfected as described in A with Shp-1 and the HA-tagged PIPKI constructs indicated (I483x, the codon for residue 483 of PIPKI661 was mutated into a stop codon, resulting in trunca- tion of the C terminus from that residue). The immunoprecipitates were then analyzed by Western blot as indicated. C, GST pull-down of PIPKI661 by Shp-1 truncations. T7-tagged PIPKI661 was pulled down by the GST-tagged Shp-1 constructs indicated. Bound PIPKI661 was detected by immunoblot with anti-T7 antibody. GST-tagged Shp-1 constructs were as follows: WT, wild type Shp-1; SH2, truncation of the Shp-1 C terminus from residue 278; PPase, truncation of the Shp-1 N terminus up to residue 300; GST, GST only. D, GST pull-down of Shp-1 C-terminal truncations. T7-tagged Shp-1 constructs were pulled down with GST-PIPKI661 and detected as described in C. C-terminal Shp-1 truncations: Shp-1 560x, C-terminal truncation of residues 560–597 by mutation of residue 560 into a stop codon; Shp-1 529x, C-terminal truncation of residues 529–597 in the same manner. Wild type T7- tagged Shp-2 (Shp-2) was used as an additional control.

Article Snippet: Horseradish peroxidase-conjugated anti-GST antibody was purchased from Amersham Biosciences, and horseradish peroxidase-conjugated anti-T7 an- tibody was obtained from Novagen.

Techniques: Transfection, Construct, Immunoprecipitation, Bioprocessing, Western Blot, Residue, Mutagenesis, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Sensory Denervation Causes Epithelial Ferroptosis and Delayed Healing in Mice

doi: 10.1167/iovs.66.6.28

Figure Lengend Snippet: Nerve degeneration induces the ferroptosis of corneal epithelial cells. ( A ) The corneal epithelial defect of surgical denervated mice was observed with slit-lamp and fluorescein sodium staining. ( B ) Corneal nerve degeneration was detected by whole-mount βIII-tubulin staining, and the percentage of nerve-covered area was analyzed by ImageJ. ( C ) Corneal sensation was measured using a Cochet–Bonnet esthesiometer. ( D ) Intracellular GSH was detected by monochlorobimane staining and analyzed by ImageJ. ( E ) The levels of Gpx4 , Gstm1 , Gsta2 , Gsta3 , and Slc7a11 were analyzed by quantitative PCR. ( F – H ) The results of immunofluorescence in the expression of GPX4 and GSTM1 proteins in the corneal epithelium of the denervated mice. The expression of GPX4 and GSTM1 in the corneal epithelium of surgical corneal denervated mice (F) and patients with neurotrophic keratitis ( G ) was detected by immunofluorescence staining, and the fluorescence intensity was quantified by ImageJ ( H ). ( I ) The mitochondrial morphology of corneal epithelium was observed by transmission electron microscopy after surgical corneal denervation. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Then, the sections were incubated with GPX4 antibodies (DF6701; Affinity Biosciences, Changzhou, China), GSTM1 antibodies (12412-1-AP; Proteintech, Rosemont, IL, USA), p53 antibodies (10442-1-AP; Proteintech), p-AKT antibody (4060; Cell Signaling Technology, Danvers, MA, USA), or p-mTOR antibodies (ab109268; Abcam, Cambridge, UK) overnight at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Immunofluorescence, Expressing, Fluorescence, Transmission Assay, Electron Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Sensory Denervation Causes Epithelial Ferroptosis and Delayed Healing in Mice

doi: 10.1167/iovs.66.6.28

Figure Lengend Snippet: TRPV1 + sensory degeneration triggers the ferroptosis of corneal epithelial cells. ( A ) The ocular surface of capsaicin (Caps)-treated mice was observed with slit-lamp and fluorescein sodium staining. ( B ) Corneal nerve degeneration was detected by βIII-tubulin staining and the percentage of nerve-covered area was analyzed by ImageJ. ( C ) Corneal sensation was measured using a Cochet–Bonnet esthesiometer. ( D ) Intracellular GSH was detected by monochlorobimane staining and analyzed by ImageJ. ( E ) The levels of Gpx4 , Gstm1 , Gsta2 , Gsta3 , and Slc7a11 were analyzed by quantitative PCR. ( F ) The expression of GPX4 and GSTM1 in the corneal epithelium of capsaicin-treated mice was detected by immunofluorescence staining and quantified by ImageJ. ( G ) The mitochondrial morphology of corneal epithelium was observed by transmission electron microscopy after capsaicin treatment. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Then, the sections were incubated with GPX4 antibodies (DF6701; Affinity Biosciences, Changzhou, China), GSTM1 antibodies (12412-1-AP; Proteintech, Rosemont, IL, USA), p53 antibodies (10442-1-AP; Proteintech), p-AKT antibody (4060; Cell Signaling Technology, Danvers, MA, USA), or p-mTOR antibodies (ab109268; Abcam, Cambridge, UK) overnight at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Transmission Assay, Electron Microscopy