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

Journal: The Journal of Clinical Investigation

Article Title: Aggregation of scaffolding protein DISC1 dysregulates phosphodiesterase 4 in Huntington’s disease

doi: 10.1172/JCI85594

Figure Lengend Snippet: (A) Soluble DISC1 but not soluble PDE4B levels in supernatant fractions (Sup) were reduced in cerebral cortex (top) and striatum (bottom) from 12-week-old R6/2 mice, compared with those from WT mice. Total DISC1 and PDE4B levels in total homogenates (Total) were not changed between WT and R6/2 mice. β-Actin is a loading control. An m317C anti-DISC1 antibody was used to detect DISC1, and a negative control with rabbit secondary antibody alone is also shown. Data represent mean + SEM. **P < 0.01, ***P < 0.001; unpaired 2-tailed t test. n = 3 per group. (B) The amount of DISC1-PDE4B complex in 12-week-old R6/2 mouse brains was decreased compared with that in WT brains. Immunoprecipitation was performed by an anti–pan-PDE4B antibody, followed by immunoblotting with anti-DISC1 antibody (D27). GAPDH is a loading control and used for normalization of the IP data. Data represent mean + SEM. **P < 0.01; unpaired 2-tailed t test. n = 5 per group. (C) Exogenous addition of HTT513Q82, but not HTT513Q18, significantly reduced DISC1-PDE4B interactions in HEK293T cells. Lysates of the cells overexpressing GFP alone (–), HTT513Q18-GFP (Q18), or HTT513Q82-GFP (Q82) were added to lysates of the cells overexpressing DISC1-Myc and PDE4B1-HA. Mixtures were processed for immunoprecipitation with an anti–c-Myc antibody, followed by immunoblotting. Data represent mean + SEM (3 independent sample sets). *P < 0.05, **P < 0.01; unpaired 2-tailed t test.

Article Snippet: The following commercial or published antibodies were also used: mouse monoclonal antibodies against HA, Myc, and GFP tags (Nacalai Tesque), against mouse DISC1 (2B3) ( 59 ), and against β-actin (Abcam); rabbit polyclonal antibodies against HA, Myc, and GFP tags (MBL), against mouse DISC1 (mEx3, refs. 59 – 63 ; and D27, refs. 63 – 66 ), against human DISC1 (14F2) ( 57 ), and against HTT (EM48 and MAB2144) (Millipore); and a sheep polyclonal anti–pan-PDE4B antibody ( 41 ).

Techniques: Control, Negative Control, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: Leafhopper salivary vitellogenin mediates virus transmission to plant phloem

doi: 10.1038/s41467-023-43488-5

Figure Lengend Snippet: a GST catalyzing the reaction of GSH and consuming H 2 O 2 . b Y2H assays showing the interaction of NcVg2 with OsGSTF12. +, positive control; –, negative control. QDO, SD/-Trp-Leu-His-Ade medium. c BiFC assays showing the interaction of NcVg2 with OsGSTF12 in N. benthamiana leaf cells. –, negative control. Bars, 100 μm. d Immunofluorescence microscopy demonstrating the colocalization of NcVg and OsGSTF12 in rice phloem exposed to leafhoppers. Panels i to ii (merged) are enlarged images of the boxed areas in d . AS, air space; P, phloem; PV, pitted vessel; ST, sieve tube. Bars, 10 μm. e , f The transient expression of OsGSTF12 increasing H 2 O 2 accumulation and activating GST-catalyzing reactions in N. benthamiana . The leaves transiently expressing OsGSTF12 e were tested for GST activity and GSH and GSSG content f . Bars, 200 μm. g OsGSTF12 expression in rice seedlings induced by leafhopper feeding, as determined by western blot assays. OsGSTF12- and histone H3-specific antibodies were used to detect proteins in western blot assays. h GST-catalyzing reactions in rice seedlings induced by leafhopper feeding, as determined by GST activity and GSH and GSSG content. Nf, non-feeding. i , j Exposure to viruliferous leafhopper improving OsGSTF12 expression and promoting GST catalyzing reaction, as determined by western blot i , activity of GST, contents of GSH and GSSG j . The proteins were detected by using OsGSTF12- or histone H3-specific antibody in western blot assays. Data in h , j , and l are shown from 1 rice seedling exposed or not exposed to 30 nonviruliferous or viruliferous leafhoppers. k , l Knockdown of NcVg expression in nonviruliferous or viruliferous leafhoppers decreasing OsGSTF12 expression and GST-catalyzing reactions. Western blot assays were used to detect proteins using OsGSTF12- and histone H3-specific antibodies. Data in f , g , h , i , j , k and l represent at least 3 biological replicates. Means ( ± SD) in c , d , f , h , j and l are shown and analyzed using two-tailed t -test. V-, nonviruliferous leafhoppers, V + , viruliferous leafhoppers.

Article Snippet: Mouse monoclonal antibodies against 6×His tag, GST, and GFP were purchased from Transgene Biotech (HT501, HT601, and HT801).

Techniques: Positive Control, Negative Control, Immunofluorescence, Microscopy, Expressing, Activity Assay, Western Blot, Knockdown, Two Tailed Test

Journal: Nature Communications

Article Title: Leafhopper salivary vitellogenin mediates virus transmission to plant phloem

doi: 10.1038/s41467-023-43488-5

Figure Lengend Snippet: a Knockout of OsGSTF12 reducing GST catalyzing reaction in rice plants exposed or not exposed to leafhoppers, as demonstrated by GST activity and contents of GSH and GSSG. b Knockout of OsGSTF12 increasing H 2 O 2 burst and metabolism in rice plants exposed or not exposed to leafhoppers, as indicated by content of H 2 O 2 and MDA, as well as activity of CAT and POD. c Knockout of OsGSTF12 increasing the number of feeding holes in leaves exposed to leafhoppers, as detected by DAB or H2DCFDA staining. One leaf of rice seedling of WT or OsGSTF12-KO lines exposed to 5 nonviruliferous leafhoppers for 12 h was tested. d Knockout of OsGSTF12 increasing difficulty of leafhoppers feeding, as determined by EPG technique. Each nonviruliferous leafhopper was continuously and electrically recorded during a 3-hour feeding period. Means ( ± SD) represent 13 valid biological replicates. e Overexpression of NcVg2 increasing OsGSTF12 expression in rice plants exposed or not exposed to leafhoppers, as determined by western blot assays using OsGSTF12-, NcVg-, or histone H3-specific antibody. f Overexpression of NcVg2 promoting GST catalyzing reaction in rice plants exposed or not exposed to leafhoppers, as demonstrated by GST activity and contents of GSH and GSSG. Data in a , b , e , and f are shown from 1 rice seedling exposed or not exposed to 30 nonviruliferous leafhoppers. Means ( ± SD) in a – d and f represent at least 3 replicates, and are analyzed using two-tailed t -test. Ns, not significant.

Article Snippet: Mouse monoclonal antibodies against 6×His tag, GST, and GFP were purchased from Transgene Biotech (HT501, HT601, and HT801).

Techniques: Knock-Out, Activity Assay, Staining, Over Expression, Expressing, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Leafhopper salivary vitellogenin mediates virus transmission to plant phloem

doi: 10.1038/s41467-023-43488-5

Figure Lengend Snippet: a Overexpression of NcVg2 promoting RDV release, while knockout of OsGSTF12 inhibiting RDV release, as indicated by western blot assays using NcVg-, P8-, OsGSTF12-, or histone H3-specific antibody. The relative intensity of the bands is shown. b Viruliferous leafhoppers feeding caused the lowest levels of H 2 O 2 burst and metabolism in NcVg2-OE lines compared to WT or OsGSTF12-KO lines. c Exposure of NcVg2-OE lines to viruliferous leafhoppers causing the lowest number of feeding holes compared to feeding on WT or OsGSTF12-KO lines, as detected by DAB or H2DCFDA staining, respectively. One leaf of rice seedling of WT, NcVg2-OE, or OsGSTF12-KO lines exposed to 5 viruliferous leafhoppers for 12 h was tested. d Exposure of NcVg2-OE lines to viruliferous leafhoppers causing the most active GST catalyzing reaction in NcVg2-OE lines, compared to WT or OsGSTF12-KO lines, as demonstrated by GST activity, GSH and GSSG contents. Data are shown from 1 leaf of WT, NcVg2-OE, or OsGSTF12-KO lines exposed to 50 viruliferous leafhoppers. e NcVg2-OE lines beneficial to RDV transmission by leafhoppers, as indicated by the transmission rate. Data are shown from 50 viruliferous leafhoppers individually feeding on 1 rice seedling of WT, NcVg2-OE, or OsGSTF12-KO lines. f The transient expression of NcVg2 significantly suppressing H 2 O 2 accumulation, inducing GST activity and reducing GSH content of N. benthamiana . The leaves separately expressed NcVg1 to NcVg4 were tested for the H 2 O 2 contents, GST activity and GSH content. g Injection of rice seedlings with His-Vg2 significantly reducing contents of H 2 O 2 , and increasing GST-catalyzing reaction in rice plants. All data represent at least 3 biological replicates. Means ( ± SD) in b, c, d, e, f , and g are analyzed using two-tailed t -test. Ns, not significant.

Article Snippet: Mouse monoclonal antibodies against 6×His tag, GST, and GFP were purchased from Transgene Biotech (HT501, HT601, and HT801).

Techniques: Over Expression, Knock-Out, Western Blot, Staining, Activity Assay, Transmission Assay, Expressing, Injection, Two Tailed Test

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