Journal: Redox Biology

Article Title: Porcine reproductive and respiratory syndrome virus nsp4 positively regulates cellular cholesterol to inhibit type I interferon production

doi: 10.1016/j.redox.2021.102207

Figure Lengend Snippet: PRRSV infection activates the PP2A–HMGCR pathway. (A) Model of cholesterol synthesis via the AMPK–HMGCR and PP2A–HMGCR pathways. Level of p-HMGCR (inactive form) was enhanced by AMPK, resulting in the downregulation of cholesterol synthesis, but reduced by PP2A, resulting in the upregulation of cholesterol synthesis. (B – D) PAMs or PK-15 CD163 cells infected with PRRSV (MOI = 1.0) were harvested at 12, 24, and 36 hpi. The expression levels of total HMGCR and phosphorylated HMGCR (p-HMGCR) (B) , total AMPK and p-AMPK (C) , or total PP2Ac and p-PP2Ac (D) were analyzed with western blotting. PRRSV infection was verified by detecting the expression of viral N protein with anti-N antibody. The β-actin was used as the protein loading control.

Article Snippet: Mouse or rabbit monoclonal antibodies (mAbs) directed against Flag, HA, GFP, and β-actin were purchased from Medical and Biological Laboratories (MBL, Japan).

Techniques: Infection, Expressing, Western Blot, Control

Journal: Redox Biology

Article Title: Porcine reproductive and respiratory syndrome virus nsp4 positively regulates cellular cholesterol to inhibit type I interferon production

doi: 10.1016/j.redox.2021.102207

Figure Lengend Snippet: PRRSV nsp4 interacts with PP2A. (A) PP2A consists of three subunits, including a structural subunit (scaffolding subunit; A subunit, PR65α), a regulatory subunit (B subunit), and a catalytic subunit (PP2Ac, C subunit). (B, C) HEK-293T cells were cotransfected with expression vector encoding Flag-tagged PR65α or PP2Ac and vector encoding HA-tagged PRRSV nsp4. The cells were lysed at 36 h post transfection and immunoprecipitated with anti-Flag (B) or anti-HA (C) antibody. Whole-cell lysates (WCLs) and immunoprecipitation (IP) complexes were analyzed with immunoblotting using anti-Flag, anti-HA, or anti-β-actin antibody. (D) PK-15 CD163 cells were infected with PRRSV (MOI = 1.0). The cells were lysed at 36 hpi and immunoprecipitated with anti-nsp4 antibody. WCLs and IP complexes were analyzed with immunoblotting using anti-nsp4, anti-PR65α, anti-PP2Ac, or anti-β-actin antibody.

Article Snippet: Mouse or rabbit monoclonal antibodies (mAbs) directed against Flag, HA, GFP, and β-actin were purchased from Medical and Biological Laboratories (MBL, Japan).

Techniques: Scaffolding, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Infection

Journal: Redox Biology

Article Title: Porcine reproductive and respiratory syndrome virus nsp4 positively regulates cellular cholesterol to inhibit type I interferon production

doi: 10.1016/j.redox.2021.102207

Figure Lengend Snippet: PRRSV nsp4 domain I (amino acids 1–80) interacts with the structural subunit (PR65α) and catalytic subunit (PP2Ac) of PP2A. (A) Schematic diagram of nsp4 truncations. (B – E) HEK-293T cells were co-transfected with expression vectors encoding Flag-tagged PR65α or PP2Ac and EGFP-tagged nsp4, nsp4 domain I, nsp4 domain II, or nsp4 domain III. The cells were lysed at 36 h post transfection and immunoprecipitated with an anti-Flag antibody (B, D) or an anti-GFP antibody (C, E) . The WCLs and IP complexes were analyzed with immunoblotting using anti-Flag, anti-EGFP, or anti-β-actin antibody.

Article Snippet: Mouse or rabbit monoclonal antibodies (mAbs) directed against Flag, HA, GFP, and β-actin were purchased from Medical and Biological Laboratories (MBL, Japan).

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric G?s Protein

doi: 10.1091/mbc.E04-06-0446

Figure Lengend Snippet: Overexpression of Gαs-GFP promotes degradation of Texas Red. (A) Cos7 cells transfected with pGαs-GFP or control vector were incubated with Texas Red EGF for 10 min and chased for 30 or 60 min. Cells expressing Gαs-GFP (traced in white) and those expressing control vector showed similar levels of Texas Red EGF at 0-min chase. However, after 30- or 60-min chase there is considerably less Texas Red EGF remaining in cells expressing Gαs-GFP. (B) Semiquantitative representation of the data shown in A. In cells transfected with control vector ∼30% of the Texas Red EGF is degraded at 30 min and 80% by 60 min, whereas in cells expressing Gαs-GFP ∼70% are degraded at 30 min and ∼95% at 60 min. Average integrated intensity of Texas Red EGF pixels per cell were measured as described in Materials and Methods. Data are expressed as the mean ± SE of three experiments.

Article Snippet: Other antibodies were obtained from the following sources: monoclonal antibodies (mAbs) against actin and FLAG (M2) (Sigma-Aldrich), myc (Cell Signaling Technology, Beverly, MA), and GFP (BD Biosciences Clontech, Palo Alto, CA), and polyclonal antibodies against EGF receptor (Santa Cruz Biotechnology, Santa Cruz, CA) and GFP and Hrs (Alexis Biochemicals, San Diego, CA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Incubation, Expressing

Journal:

Article Title: Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric G?s Protein

doi: 10.1091/mbc.E04-06-0446

Figure Lengend Snippet: Interaction between Hrs and Gαs. (A) In vitro-translated, 35S-labeled Gαs binds to GST-Hrs but not to GST alone. GST-Hrs and GST proteins (∼75 μg each) immobilized on glutathione beads were incubated with in vitro-translated, [35S]Gαs as in Figure 4. Input equals 3% of total in vitro translation product. (B) Endogenous Gαs from rat brain lysates binds to GST-Hrs but not to GST. GST-Hrs and GST immobilized on glutathione beads were incubated with rat brain lysates (∼5 mg). Bound proteins were immunoblotted with anti-Gαs IgG. Input equals 3% of total brain lysate. (C) Myc-Hrs coimmunoprecipitates with Gαs-GFP (lane 4). Lysates (lanes 1 and 2) from HEK293 cells transfected with Gαs-GFP or GFP together with myc-Hrs were immunoprecipitated with anti-GFP, followed by immunoblotting with anti-myc and anti-GFP antibodies. (D) Gαs and Hrs are found in approximately equal amounts in both membrane (P100, lane 2) and cytosolic (S100, lane 1) fractions. Gαs coimmunoprecipitates with myc-Hrs predominantly (>95%) from membrane fractions (lane 4, bottom). Very little Gαs is coprecipitated with myc-Hrs from the cytosolic fraction (lane 3, bottom). Cytosolic (S100, lane 1) and membrane (P100, lane 2) fractions prepared from HEK293 cells transfected with Gαs and myc-Hrs were immunoprecipitated with anti-myc (myc, lanes 3 and 4) or control (ctrl, lanes 5 and 6) mouse IgGs, followed by immunoblotting with anti-Gαs and anti-myc antibodies.

Article Snippet: Other antibodies were obtained from the following sources: monoclonal antibodies (mAbs) against actin and FLAG (M2) (Sigma-Aldrich), myc (Cell Signaling Technology, Beverly, MA), and GFP (BD Biosciences Clontech, Palo Alto, CA), and polyclonal antibodies against EGF receptor (Santa Cruz Biotechnology, Santa Cruz, CA) and GFP and Hrs (Alexis Biochemicals, San Diego, CA).

Techniques: In Vitro, Labeling, Incubation, Transfection, Immunoprecipitation, Western Blot

Journal:

Article Title: Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric G?s Protein

doi: 10.1091/mbc.E04-06-0446

Figure Lengend Snippet: Colocalization of Gαs-GFP with myc-Hrs on early endosomes. (A–C) In Cos7 cells transfected with Gαs-GFP alone, Gαs is distributed on the plasma membrane (arrow, A) and on small vesicular structures (arrowheads, A). Hrs is distributed on early endosomes throughout the cell (B). Merged image (yellow) shows occasional overlap in the vesicular distribution of Gαs-GFP and Hrs (arrowheads and inset, C). (D–I) In cells transfected with Myc-Hrs, which promotes formation of large, clustered endosomes Gαs-GFP is distributed on the plasma membrane (arrow, D) and on the enlarged endosomes (arrowheads and inset, D and G). Myc-Hrs (arrowheads and inset, E) and Rab5 (arrowheads and inset, H) are also present on these endosomes. Gαs-GFP colocalizes (yellow) with Myc-Hrs (arrowheads and inset, F) and Rab5 (arrowhead and inset, I). Cos7 cells were transfected with Gαs-GFP alone (A–C) or together with Myc-Hrs (D–I) and permeabilized with saponin before fixation to release the cytosolic proteins and facilitate the detection of membrane-associated pools of Gαs and Hrs. Cells were then fixed with 3% PFA, permeabilized, and double labeled with mouse anti-GFP (A, D, and G), anti-Hrs (B), or anti-myc (E) IgG or rabbit anti-rab5 (H) IgG and analyzed by deconvolution immunofluorescence microscopy. Bar, 2 μm.

Article Snippet: Other antibodies were obtained from the following sources: monoclonal antibodies (mAbs) against actin and FLAG (M2) (Sigma-Aldrich), myc (Cell Signaling Technology, Beverly, MA), and GFP (BD Biosciences Clontech, Palo Alto, CA), and polyclonal antibodies against EGF receptor (Santa Cruz Biotechnology, Santa Cruz, CA) and GFP and Hrs (Alexis Biochemicals, San Diego, CA).

Techniques: Transfection, Labeling, Immunofluorescence, Microscopy

Journal:

Article Title: Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric G?s Protein

doi: 10.1091/mbc.E04-06-0446

Figure Lengend Snippet: Immunogold localization of Hrs and Gαs in HEK293 cells. (A). Gαs-GFP (10-nm gold) is localized to numerous coated tubules (arrowheads) and endosomes (asterisks). (B and C). Myc-Hrs (5-nm gold, arrows) and Gαs-GFP (10-nm gold) colocalize in coated domains of early endosomes (asterisks). HEK293 cells transfected with myc-Hrs and Gαs-GFP were fixed either in 4% PFA (A) or a mixture of 4% PFA and 0.2% glutaraldehyde (B and C) and prepared for ultrathin cryosectioning as described in Materials and Methods. Ultrathin cryosections were labeled with anti-myc mAb (5-nm gold) and polyclonal anti-GFP (10-nm gold). Bar, 100 nm.

Article Snippet: Other antibodies were obtained from the following sources: monoclonal antibodies (mAbs) against actin and FLAG (M2) (Sigma-Aldrich), myc (Cell Signaling Technology, Beverly, MA), and GFP (BD Biosciences Clontech, Palo Alto, CA), and polyclonal antibodies against EGF receptor (Santa Cruz Biotechnology, Santa Cruz, CA) and GFP and Hrs (Alexis Biochemicals, San Diego, CA).

Techniques: Transfection, Labeling

Journal:

Article Title: Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric G?s Protein

doi: 10.1091/mbc.E04-06-0446

Figure Lengend Snippet: Colocalization of Gαs with RGS-PX1 on early endosomes. GFP-RGS-PX1 is found on endosomes (arrowheads and insets, A and D) and partially colocalizes with Gαs-WT (arrowheads and inset, B and C) on endosomes loaded with Texas Red EGF (arrowheads and inset, E and F). Cos7 cells were transfected with GFP-RGS-PX1 and Gαs-WT. In D–F, cells also were incubated with Texas Red EGF for 15 min at 37°C. Cells were permeabilized with saponin, fixed with 3% PFA, double labeled with mouse anti-GFP mAb (A and D), and rabbit anti-Gαs (B) IgG, and analyzed as described in Figure 6. Bar, 2 μm.

Article Snippet: Other antibodies were obtained from the following sources: monoclonal antibodies (mAbs) against actin and FLAG (M2) (Sigma-Aldrich), myc (Cell Signaling Technology, Beverly, MA), and GFP (BD Biosciences Clontech, Palo Alto, CA), and polyclonal antibodies against EGF receptor (Santa Cruz Biotechnology, Santa Cruz, CA) and GFP and Hrs (Alexis Biochemicals, San Diego, CA).

Techniques: Transfection, Incubation, Labeling