Journal: The Journal of biological chemistry

Article Title: Protein kinase A-dependent translocation of Hsp90 alpha impairs endothelial nitric-oxide synthase activity in high glucose and diabetes.

doi: 10.1074/jbc.M608985200

Figure Lengend Snippet: FIGURE 2. High glucose promoted PKA-dependent phosphorylation of Hsp90. A, Hsp90 was immunoprecipitated (IP) with an anti-Hsp90 anti- body from total lysates (using 1 107 cells/sample) as described under “Experimental Procedures.” Immunoprecipitates were prepared in parallel with non-immune IgG. The resulting samples were subjected to a phos- phospecific PKA substrate Western blot (top panel). The stripped membrane was reprobed with a Hsp90 antibody (bottom panel). The ratio of the signal from the bottom and top panels is presented. The results show that there was 2.3-fold more phosphorylated Hsp90 in samples prepared from the HG- treated cells as compared with those cultured in normal glucose. In three independent experiments, the fold difference was 2.2 0.2. B, same as A except the HG-treated cells were exposed to the PKA inhibitor PKI (0.5 mol/ liter) for 12 h prior to lysis. The PKI treatment reduced the amount of Hsp90 recognizedbythephospho-PKAsubstrateantibodyby2.6-fold.Inthreeinde- pendent experiments, the average fold difference was 2.7 0.2.

Article Snippet: The following antibodies were obtained from commercial sources: phospho-(Ser/Thr) PKA substrate antibody (number 9621, Cell Signaling Technology, Beverly, MA); anti-FLAG M2 monoclonal antibody (Sigma), and anti-eNOS mouse monoclonal antibody (BD Transduction Laboratories); goat anti-Hsp90 polyclonal antibody and non-immune goat IgG (Santa Cruz Biotechnology); horseradish peroxidase-conjugated goat antirabbit antibodies (AmershamBiosciences); and fluorescein isothiocyante-conjugated AffiniPure sheep anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Membrane, Cell Culture, Lysis

Journal: The Journal of biological chemistry

Article Title: Protein kinase A-dependent translocation of Hsp90 alpha impairs endothelial nitric-oxide synthase activity in high glucose and diabetes.

doi: 10.1074/jbc.M608985200

Figure Lengend Snippet: FIGURE 4. Phosphorylated Hsp90 was predominately on the cell sur- face. PAECs were treated with HG (30 mmol/liter) for 2 weeks. Hsp90 was immunoprecipitated (IP) with an anti-Hsp90 antibody from the cell surface (using 3 107 cells/sample) or from total lysates (using 0.5 107 cells/sam- ple) as described under “Experimental Procedures.” Immunoprecipitates were prepared in parallel with non-immune IgG. The resulting samples were subjected to a phosphospecific PKA substrate Western blot (top panel). The stripped membrane was reprobed with a Hsp90 antibody (bottom panel). Theratioofthesignalfromthebottomandtoppanelsispresented.Theresults showed that there was 13.6-fold more phosphorylated Hsp90 in samples prepared from the cell surface as compared with total cell lysates. In three independent experiments, the average -fold difference was 11.8 1.5.

Article Snippet: The following antibodies were obtained from commercial sources: phospho-(Ser/Thr) PKA substrate antibody (number 9621, Cell Signaling Technology, Beverly, MA); anti-FLAG M2 monoclonal antibody (Sigma), and anti-eNOS mouse monoclonal antibody (BD Transduction Laboratories); goat anti-Hsp90 polyclonal antibody and non-immune goat IgG (Santa Cruz Biotechnology); horseradish peroxidase-conjugated goat antirabbit antibodies (AmershamBiosciences); and fluorescein isothiocyante-conjugated AffiniPure sheep anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Membrane

Journal: The Journal of biological chemistry

Article Title: Protein kinase A-dependent translocation of Hsp90 alpha impairs endothelial nitric-oxide synthase activity in high glucose and diabetes.

doi: 10.1074/jbc.M608985200

Figure Lengend Snippet: FIGURE 8. DM increased phosphorylation of Hsp90 at a PKA consen- sus site and decreased the Hsp90eNOS complex. A, lysates prepared from the aortic endothelium of five diabetic or age-matched control rats were pooled, and analyzed by Western blotting using a phosphospecific PKA substrate antibody (top panel) and a RasGAP antibody (lower panel). B, the lysates described in A were immunoprecipitated (IP) with a Hsp90 antibody or non-immune IgG, and the resulting samples were analyzed by Western blot using a phosphospecific PKA substrate antibody (top panel). The stripped membrane was reprobed with a Hsp90 antibody (bottom panel). Quantification of Western blots showed a 2.2 0.2-fold increase in the amount of phospho-Hsp90 in the diabetic endothelium in three independent experiments. C, the Western blot described in B was reprobed using an anti-eNOS antibody. DM reduced the association of eNOS with Hsp90 in the endothelium of the diabetic rat aorta by 2.4 0.2-fold com- pared with the controls. The differences in panels B and C were statistically significant.

Article Snippet: The following antibodies were obtained from commercial sources: phospho-(Ser/Thr) PKA substrate antibody (number 9621, Cell Signaling Technology, Beverly, MA); anti-FLAG M2 monoclonal antibody (Sigma), and anti-eNOS mouse monoclonal antibody (BD Transduction Laboratories); goat anti-Hsp90 polyclonal antibody and non-immune goat IgG (Santa Cruz Biotechnology); horseradish peroxidase-conjugated goat antirabbit antibodies (AmershamBiosciences); and fluorescein isothiocyante-conjugated AffiniPure sheep anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Phospho-proteomics, Control, Western Blot, Immunoprecipitation, Membrane

Journal: Journal of neurochemistry

Article Title: Restricted transport of anti-transferrin receptor antibody (OX26) through the blood-brain barrier in the rat.

doi: 10.1046/j.1471-4159.2001.00541.x

Figure Lengend Snippet: Fig. 2 Changes in plasma concentration of the OX26 and Ni-IgG2a with time after injection expressed as % ID/mL plasma ^ SD in adult (a) and P15 (b) rats. Initially, the concentration of OX26 decreases markedly to a signi®cantly lower concentration than Ni-IgG2a, and from 2 min and onwards, the plasma concentration of OX26 and Ni-IgG2a are statistically different at any time point. The diamonds marked by an arrow represent the corresponding values of OX26 and Ni-IgG2a (identical colour) of iron-de®cient P15 rats; these values are not statistically different to the corresponding values of P15 rats having a normal iron status. Each value is the mean ^ SD of measurements from 3 to 5 rats. A, OX26; B, Ni-Ig2a.

Article Snippet: Puri®ed monoclonal mouse-anti-rat transferrin receptor IgG2a (clone OX26) and non-immune mouse IgG2a (Ni-IgG2a) were purchased from Serotec (Oxford, UK).

Techniques: Clinical Proteomics, Concentration Assay, Injection

Journal: Journal of neurochemistry

Article Title: Restricted transport of anti-transferrin receptor antibody (OX26) through the blood-brain barrier in the rat.

doi: 10.1046/j.1471-4159.2001.00541.x

Figure Lengend Snippet: Fig. 3 Accumulation of OX26 and Ni-IgG2a expressed as % ID/organ ^ SD in adult brain (a) and adult liver (b). The accumu- lation of both OX26 and Ni-IgG2a is much higher in the liver than in the brain (*). At 60 min, the accumulation of OX26 in the brain is signi®cantly higher than at 30 min post-injection. A, OX26; B, Ni-Ig2a.

Article Snippet: Puri®ed monoclonal mouse-anti-rat transferrin receptor IgG2a (clone OX26) and non-immune mouse IgG2a (Ni-IgG2a) were purchased from Serotec (Oxford, UK).

Techniques: Injection

Journal: Journal of neurochemistry

Article Title: Restricted transport of anti-transferrin receptor antibody (OX26) through the blood-brain barrier in the rat.

doi: 10.1046/j.1471-4159.2001.00541.x

Figure Lengend Snippet: Fig. 6 Accumulation of OX26 and Ni-IgG2a in CSF of adult and P15 rats expressed as % ID/mL CSF 1 SD (a) or Vd expressed as mL plasma/mL CSF 1 SD (b). (a) The uptake of both proteins are signi®cantly higher in the P15 rat than in the adult (*). OX26 accu- mulates to a signi®cantly higher degree than Ni-IgG2a in both the adult and P15 rat (o). (b) When expressed in terms of Vd, OX26 accumulation is greater than Ni-IgG2a at both ages (o). The Vd values of OX26 and Ni-IgG2a are not statistically different.

Article Snippet: Puri®ed monoclonal mouse-anti-rat transferrin receptor IgG2a (clone OX26) and non-immune mouse IgG2a (Ni-IgG2a) were purchased from Serotec (Oxford, UK).

Techniques: Clinical Proteomics