Journal: International Journal of Molecular Sciences

Article Title: Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia

doi: 10.3390/ijms19092518

Figure Lengend Snippet: Effect of stability to dilution, pH, and thermostability of recombinant G6PD::6PGL from G. lamblia. ( A ) The enzyme was incubated at the indicated protein concentrations for two hours at 37 °C in 50 mM phosphate buffer at pH 8.7. At the indicated time, the residual activity was measured with 1 µg/mL of sample for each case. ( B ) Effect of pH on the activity of G6PD::6PGL protein. The residual activity was tested by measuring the activity under standard conditions at each pH value. Buffers used were MES (pH 6.0–6.75), HEPES (pH 6.75–8.0), Tris (pH 8.0–9.0), and Glycine (pH 9.0–10). The data represent the mean ± SD from three independent measurements. ( C ) The protein thermostability was studied by measuring the residual activities after the enzymes were incubated at different temperatures (37–60 °C) for 20 min in 50 mM Tris buffer at pH 8.75.

Article Snippet: The G6PD::6PGL protein and the gel filtration standards (Biorad, Hercules, CA, USA) were applied to a Sephacryl 100 (16/60) gel filtration column (GE Healthcare) that had been pre-equilibrated with 50 mM Tris buffer at pH 7.85 and was coupled to the AKTA pure FPLC system (GE Healthcare) using the same buffer as the mobile phase with a flow rate of 0.5 mL·min −1 .

Techniques: Recombinant, Incubation, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia

doi: 10.3390/ijms19092518

Figure Lengend Snippet: Stability analysis of recombinant G6PD::6PGL from G. lamblia . ( A ) Thermal stability. Changes in the circular dichroism (CD) signal at 222 nm were monitored as the temperature increased from 20 °C to 80 °C. ( B ) Stability analysis of protein in the presence of Gdn-HCl (0 to 1 M). The protein was incubated at 0.2 mg/mL in 50 mM Tris buffer pH 8.75 in the presence of the indicated concentrations of Gdn-HCl for 2 h at 37 °C and subsequently the enzymatic activity was measured. Residual activity for G6PD was expressed as a percentage of the activity for the same sample measured at 25 °C, without Gdn-HCl, and were diluted immediately before use. The experiments were performed in triplicate.

Article Snippet: The G6PD::6PGL protein and the gel filtration standards (Biorad, Hercules, CA, USA) were applied to a Sephacryl 100 (16/60) gel filtration column (GE Healthcare) that had been pre-equilibrated with 50 mM Tris buffer at pH 7.85 and was coupled to the AKTA pure FPLC system (GE Healthcare) using the same buffer as the mobile phase with a flow rate of 0.5 mL·min −1 .

Techniques: Recombinant, Incubation, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia

doi: 10.3390/ijms19092518

Figure Lengend Snippet: Structural analysis of G6PD::6PGL by intrinsic fluorescence. ( A ) Intrinsic fluorescence spectra in the presence of Gdn-HCl. ( B ) Effect on the intrinsic fluorescence at different concentrations of Gdn-HCl. The protein was incubated at 0.1 mg/mL in 50 mM Tris buffer pH 8.75 in the presence of the indicated concentrations of Gdn-HCl for two hours at 37 °C and subsequently the intrinsic fluorescence was measured. The experiments were performed in triplicate and the standard errors were less than 4%. Values obtained from buffer containing Gdn-HCl without protein were subtracted from the measurements of G6PD::6PGL protein.

Article Snippet: The G6PD::6PGL protein and the gel filtration standards (Biorad, Hercules, CA, USA) were applied to a Sephacryl 100 (16/60) gel filtration column (GE Healthcare) that had been pre-equilibrated with 50 mM Tris buffer at pH 7.85 and was coupled to the AKTA pure FPLC system (GE Healthcare) using the same buffer as the mobile phase with a flow rate of 0.5 mL·min −1 .

Techniques: Fluorescence, Incubation

Journal: Journal of Biological Chemistry

Article Title: RAFTK/Pyk2 Activation Is Mediated by Trans-acting Autophosphorylation in a Src-independent Manner

doi: 10.1074/jbc.m313527200

Figure Lengend Snippet: FIG. 3. Trans-acting autophosphorylation of endogenous RAFTK upon potassium depolarization in PC12 cells. A, PC12 cells were transfected with GFP-tagged WT RAFTK or KM RAFTK cDNA (4 g) using LipofectAMINE 2000 (Invitrogen). Empty vector pEGFP-C3 (V-GFP) cDNA was used as a control. After a 24-h incuba- tion, the cells were unstimulated or stimulated with KCl (60 mM, 5 min). The cells were harvested with RIPA buffer followed by immuno- precipitation (IP) with mouse anti-GFP antibodies (MBL) and immu- noblotting (IB) with specific antibodies, as indicated. The phosphoryl- ation of endogenous RAFTK was examined by immunoprecipitation with rabbit anti-RAFTK antibody and immunoblotting with specific antibodies, as indicated. B, HEK 293 cells were transfected with GFP- tagged WT RAFTK or KM RAFTK cDNA (2 g) using the calcium phosphate method. Empty vector pEGFP-C3 (V-GFP) cDNA was used as a control. After 24 h of incubation, the cells were unstimulated or stimulated with KCl (60 mM, 5 min). Whole cell lysate preparation, immunoprecipitation, and immunoblotting procedures were the same as described above (for A). The phosphotyrosine ratio (pY/RAFTK or Tyr(P)402/RAFTK) demonstrates the relative amounts of phosphoryla- tion at each tyrosine- or Tyr402-specific phosphorylation in the samples.

Article Snippet: The cell lysates were prepared with modified RIPA buffer and immunoprecipitated (IP) with specific antibodies against FLAG (goat antiFLAG from Santa Cruz Biotechnology) or against GFP (mouse antiGFP antibody from MBL).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Incubation, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: RAFTK/Pyk2 Activation Is Mediated by Trans-acting Autophosphorylation in a Src-independent Manner

doi: 10.1074/jbc.m313527200

Figure Lengend Snippet: FIG. 4. Self-association of RAFTK fused with different tagging epitopes. A, HEK 293 cells were co-transfected with 0.2 g of FLAG- tagged wild type (WT-FLAG) RAFTK with various amounts of GFP- tagged wild type (WT-GFP) RAFTK, followed by immunoprecipitation (IP) with goat anti-FLAG antibody (Santa Cruz Biotechnology) and immunoblotting (IB) with mouse anti-RAFTK antibody as indicated. The percentage of GFP/FLAG demonstrates the relative amounts of differentially tagged RAFTK in the samples. B, in vitro pull-down assay using fast protein liquid chromatography affinity chromatography with anti-FLAG antibody-attached agarose beads. 2 g each of RAFTK WT- FLAG and WT-GFP cDNA constructs were used for co-transfection on each 100-mm dish of HEK 293 cells. After 48 h of transfection, the cells were harvested with modified RIPA buffer. 4 mg of whole cell lysates were loaded on the column with anti-FLAG antibody-attached agarose beads. Elutes of each fraction were resolved by gel electrophoresis and probed with mouse anti-RAFTK antibody to show the association of GFP-tagged RAFTK (WT-GFP) with FLAG-tagged RAFTK (WT- FLAG). The protein ratio (GFP/FLAG) demonstrates the relative amounts of differentially tagged RAFTK in the samples. WCL, whole cell lysates.

Article Snippet: The cell lysates were prepared with modified RIPA buffer and immunoprecipitated (IP) with specific antibodies against FLAG (goat antiFLAG from Santa Cruz Biotechnology) or against GFP (mouse antiGFP antibody from MBL).

Techniques: Transfection, Immunoprecipitation, Western Blot, In Vitro, Pull Down Assay, Fast Protein Liquid Chromatography, Affinity Chromatography, Construct, Cotransfection, Modification, Nucleic Acid Electrophoresis

Journal: Journal of Biological Chemistry

Article Title: RAFTK/Pyk2 Activation Is Mediated by Trans-acting Autophosphorylation in a Src-independent Manner

doi: 10.1074/jbc.m313527200

Figure Lengend Snippet: FIG. 6. Role of Src in RAFTK autophosphorylation. Various FLAG- or GFP-tagged RAFTK cDNA constructs were transfected into SYF or SYF Src cells with LipofectAMINE 2000 (Invitrogen). Empty vector pEGFP-C3 (A and C, Vector-GFP) or FLAG-tagged cloned pcDNA3 (B, Vector-FLAG) cDNAs were used as transfection controls. After 48 h, the cells were harvested with RIPA buffer, followed by immunoprecipitation (IP) and immunoblotting (IB) with specific anti- bodies, as indicated. A, autophosphorylation of various RAFTK con- structs in SYF or SYF Src cells. The cell lysates (500 g) were immunoprecipitated with either goat anti-FLAG antibody (for del C-

Article Snippet: The cell lysates were prepared with modified RIPA buffer and immunoprecipitated (IP) with specific antibodies against FLAG (goat antiFLAG from Santa Cruz Biotechnology) or against GFP (mouse antiGFP antibody from MBL).

Techniques: Construct, Transfection, Plasmid Preparation, Clone Assay, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: RAFTK/Pyk2 Activation Is Mediated by Trans-acting Autophosphorylation in a Src-independent Manner

doi: 10.1074/jbc.m313527200

Figure Lengend Snippet: FIG. 7. Effects of Src kinase on RAFTK-mediated paxillin phos- phorylation. SYF or SYF Src cells were co-transfected with 0.5 g of Myc-tagged paxillin (Myc-pax) cDNA and 1.5 g of various FLAG- or GFP-tagged RAFTK cDNA constructs. Empty vector pEGFP-C3 (Vec- tor-GFP) cDNA was used as a transfection control. The cell lysates were prepared with modified RIPA buffer, followed by immunoprecipitation (IP) with monoclonal anti-Myc antibody (Upstate Biotechnology Inc.) and immunoblotting (IB) with mouse phosphotyrosine-specific antibody (4G10; Upstate Biotechnology Inc.) and mouse anti-paxillin antibody (Transduction Laboratory).

Article Snippet: The cell lysates were prepared with modified RIPA buffer and immunoprecipitated (IP) with specific antibodies against FLAG (goat antiFLAG from Santa Cruz Biotechnology) or against GFP (mouse antiGFP antibody from MBL).

Techniques: Transfection, Construct, Plasmid Preparation, Control, Modification, Immunoprecipitation, Western Blot, Transduction