Journal:

Article Title: RGS4 and RGS2 Bind Coatomer and Inhibit COPI Association with Golgi Membranes and Intracellular Transport

doi:

Figure Lengend Snippet: Cofractionation of endogenous RGS proteins and COPI. (A) Endogenous RGS4 cofractionates with β-COP in NG108 cells by gel filtration. Protein molecular weight standards were loaded onto a Synchropack GPC 100 HPLC column; the elution profile is shown in the chromatograph for thyroglobulin (669,000), aldolase (150,000), and chymotrypsinogen (25,000) molecular weight standards (solid line). NG108 cytosol was loaded onto the column, and 0.5-ml fractions were collected. The entire amount of protein in peak fractions was separated on 12% gels, transferred to nitrocellulose, and probed with specific antibodies against β-COP or RGS4 as indicated. (B) Characterization of RGS2 antibody. 6His-RGS2 or -RGS4 (50 ng) was electrophoresed and immunoblotted with RGS2 antiserum. The antiserum was preincubated with either a control peptide (FLAG; left panel) or immunizing peptide (middle panel) (both at 10 μg/ml) for 30 min at room temperature before immunodetection. Right panel shows lysate from HEK293T cells (100–150 μg of protein) treated with PMA (100 ng/ml for 2 h) (lane 1) or 6His-RGS2 as a positive control (lane 2) electrophoresed and blotted with RGS2 antiserum. (C) Endogenous RGS2 cofractionates with β′-COP in HEK293T cells. The cytosolic fraction from PMA-treated HEK293T cells was loaded onto a Superdex 200 FPLC column and fractionated as in A. Molecular weight marker migrations are indicated by arrows (thyroglobulin, 669,000; γ-globulin, 158,000; and ovalbumin, 44,000). Peak protein-containing fractions were separated by SDS-PAGE and immunodetected with antibodies for β′-COP (top panel) or RGS2 (bottom panel).

Article Snippet: The cytosolic fraction (supernatant) was concentrated by ultrafiltration and loaded onto a Synchropack GPC 100 HPLC (NG108) or Superdex 200 fast performance liquid chromatography (FPLC) (HEK293T) column (Amersham Pharmacia Biotech).

Techniques: Filtration, Molecular Weight, Immunodetection, Positive Control, Marker, SDS Page

Journal:

Article Title: RGS4 and RGS2 Bind Coatomer and Inhibit COPI Association with Golgi Membranes and Intracellular Transport

doi:

Figure Lengend Snippet: RGS4 inhibits secretion of placental alkaline phosphatase in HEK293T cells. Cells were transfected with plasmids encoding SEAP cytomegalovirus–β-galactosidase and a control vector or various HA–RGS4 plasmids. Forty-eight hours after transfection, medium was washed out and replaced with fresh medium for 4 h. Alkaline phosphatase was measured from the supernatant and cell lysate as luciferase activity and divided by β-galactosidase values to obtain normalized SEAP levels. The bar graph represents means ± SEM of two independent experiments (assayed in triplicate) expressed as a percentage of control vector alone (ratios of SEAP in supernatant and the sum of alkaline phosphatase in supernatant and cell lysate).

Article Snippet: The cytosolic fraction (supernatant) was concentrated by ultrafiltration and loaded onto a Synchropack GPC 100 HPLC (NG108) or Superdex 200 fast performance liquid chromatography (FPLC) (HEK293T) column (Amersham Pharmacia Biotech).

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Toxins

Article Title: Colombian Scorpion Centruroides margaritatus : Purification and Characterization of a Gamma Potassium Toxin with Full-Block Activity on the hERG1 Channel

doi: 10.3390/toxins13060407

Figure Lengend Snippet: Purification of potassium channel toxins from the venom of Centruroides margaritatus . ( A ) Venom separation by RP-FPLC resulting in 41 fractions. Fraction F23 is active on hERG1 channels and is indicated by the red arrow. The inset shows an example of the hERG1 current (black line) and the blocking effect of F23 (red line). ( B ) Re-purification of the fraction F23; the pure peptide active on hERG1 is indicated by the asterisk and corresponds to the one with MW 4792.88 Da and called CmERG1. ( C – E ) Isolation of CmERG1 by means of a three-step protocol. First, gel filtration leads to three principal fractions FI, FII and FIII ( C ). From these, fraction FII was further separated by cation-exchange chromatography into 10 sub-fractions ( D ). Toxin CmERG1 was isolated by RP-HPLC from fractions FII.6 with a retention time of 27.4 min (indicated with a star in E). In C and D, the red arrow indicates the fraction separated in the subsequent step.

Article Snippet: The venom was fractionated by means of FPLC high performance liquid chromatography model NGC chromatography System, Chrom lab Model software, and Bio Frac automatic fraction collector (Bio Rad, Hercules, CA, USA).

Techniques: Purification, Blocking Assay, Isolation, Filtration, Chromatography

Journal: Toxins

Article Title: Colombian Scorpion Centruroides margaritatus : Purification and Characterization of a Gamma Potassium Toxin with Full-Block Activity on the hERG1 Channel

doi: 10.3390/toxins13060407

Figure Lengend Snippet: Mass-spectrometry analysis of the most abundant peaks obtained by RP-FPLC.

Article Snippet: The venom was fractionated by means of FPLC high performance liquid chromatography model NGC chromatography System, Chrom lab Model software, and Bio Frac automatic fraction collector (Bio Rad, Hercules, CA, USA).

Techniques: