Journal: Journal of Biological Chemistry

Article Title: Regulation of Constitutive Cargo Transport from the trans-Golgi Network to Plasma Membrane by Golgi-localized G Protein βγ Subunits

doi: 10.1074/jbc.m110.154963

Figure Lengend Snippet: FIGURE6.InhibitionofVSV-GtransportfromTGNtothePMbyGseques- tration. A, HeLa cells were transfected with GRK2ct, GRK2ct R587Q, Golgi- GRK2ct, Golgi-GRK2ct R587Q, or PM-GRK2ct along with GFP-tsO45 VSV-G. Fol- lowingtheprotocoldescribedunder“ExperimentalProcedures,”cellswerefixed immediately before (0 min) or 120 min after the shift to 32 °C. VSV-G and GRK2ct localization was detected by immunofluorescence microscopy using anti-GFP and anti-GRK2 antibodies. VSV-G is retained in the Golgi in GRK2ct- and Golgi- GRK2ct-expressingcellsafter120-minincubationatthepermissivetemperature, compared with the cell-surface localization of VSV-G in cells expressing GRK2ct R587Q, Golgi-GRK2ct R587Q, and PM-GRK2ct. Emanating tubules (indicated by insets) were observed in GRK2ct- and Golgi-GRK2ct-expressing cells. Arrowheads indicate plasma membrane localization of VSV-G. Scale bar, 10 m. B, 100 cells were counted in 6 different experiments. The percentage of cells that show VSV-G at the PM were 30% and 20% in cells expressing GRK2ct and Golgi- GRK2ct, respectively. Statistical significance was tested using unpaired t test (*, p 0.001).

Article Snippet: Cells were then incubated with the indicated primary antibodies in blocking buffer for 1 h. Primary antibodies used included anti-TGN46 antibody (Abcam), anti-Myc antibody 9E10 (Covance), anti-GFP antibody (Rockland), anti- 2 antibody (Santa Cruz Biotechnology), anti- 1–4 antibody (Santa Cruz Biotechnology), and anti-GRK2 antibody (a generous gift from Dr. Jeffrey Benovic).

Techniques: Transfection, Immunofluorescence, Microscopy, Expressing, Clinical Proteomics, Membrane

Journal: Journal of Biological Chemistry

Article Title: Regulation of Constitutive Cargo Transport from the trans-Golgi Network to Plasma Membrane by Golgi-localized G Protein βγ Subunits

doi: 10.1074/jbc.m110.154963

Figure Lengend Snippet: FIGURE 7. Inhibition of ss-HRP secretion by Gallein and GRK2ct. HeLa cells were transfected with ss-HRP expression vector for 24 h before washing cells and then incubating another 5 h before assaying secreted HRP activity in the media. In addition, cells were transfected 24 h before the ss-HRP transfection with empty vector pcDNA3 (as control), GRK2ct, GRK2ctR587Q, Golgi-GRK2ct, or Golgi-GRK2ctR587Q, as indicated, or cells were treated with 10 M gallein and fluorescein for a total of 5.5 h before measuring secreted HRP activity as described under “Experimental Procedures.” Bars represent the mean S.D. of HRP activity in the medium from three experiments each performed in triplicate. Statistical significance compared with control (pcDNA3) was tested using unpaired t test (*, p 0.001). The lower panel shows a Western blot using cell lysates and an anti-GRK2 antibody to demonstrate similar expres- sion levels of GRK2ct, GRK2ctR587Q, Golgi-GRK2ct, and Golgi-GRK2ctR587Q.

Article Snippet: Cells were then incubated with the indicated primary antibodies in blocking buffer for 1 h. Primary antibodies used included anti-TGN46 antibody (Abcam), anti-Myc antibody 9E10 (Covance), anti-GFP antibody (Rockland), anti- 2 antibody (Santa Cruz Biotechnology), anti- 1–4 antibody (Santa Cruz Biotechnology), and anti-GRK2 antibody (a generous gift from Dr. Jeffrey Benovic).

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Activity Assay, Control, Western Blot

Journal: Circulation Research

Article Title: Exercise Protects Against Myocardial Ischemia–Reperfusion Injury via Stimulation of β 3 -Adrenergic Receptors and Increased Nitric Oxide Signaling: Role of Nitrite and Nitrosothiols

doi: 10.1161/circresaha.111.241117

Figure Lengend Snippet: Figure 2. Voluntary exercise (VE) train- ing increased the cardiac expression of CuZnSOD and altered the phos- phorylation status of AMP-activated protein kinase (AMPK) in comparison with sedentary (SED) controls. A, Rep- resentative immunoblots and densito- metric analysis of cardiac HSP70 (B), CuZnSOD (C), total AMPK and phos- phorylated AMPK at threonine residue 172 (AMPK-PThr172) (D) following 4 weeks of VE training. Values are meansSEM. *P0.05 versus SED.

Article Snippet: The following primary antibodies were used: anti-rabbit CuZnSOD, Cell Signaling; anti-rabbit AMPK-PThr172, Cell Signaling; anti-rabbit AMPK, Cell Signaling; anti-rabbit HSP70, Cell Signaling; anti-mouse eNOS, BD Transduction Laboratories; anti-rabbit eNOSSer1177, Cell Signaling; anti-rabbit eNOSThr495, Cell Signaling; anti-mouse iNOS, BD Transduction Laboratories; anti-mouse nNOS, BD Transduction Laboratories; anti-rabbit β1-adrenergic receptor, Abcam; anti-rabbit β2adrenergic receptor, Abcam; anti-chicken β3-adrenergic receptor, Abcam.

Techniques: Expressing, Comparison, Western Blot, Residue