Journal: Molecular biology of the cell

Article Title: Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

doi: 10.1091/mbc.e06-12-1120

Figure Lengend Snippet: Figure 1. Interaction between ATXN2 and the DEAD/H-box RNA helicase DDX6. (A) Schematic illustration of ATXN2. Ellipses rep- resent the polyQ-region (dark red), LSm and LSmAD domain (dark blue and light blue, respectively). Bars below display ATXN2 re- gions used in the directed yeast two-hybrid analysis. (B) Yeast two-hybrid analysis. Yeast strain L40ccua was transformed with the respective plasmids to coexpress the different LexA and AD fusion proteins as indicated. Afterward, transformants were isolated and spotted on selective media or on a membrane to analyze the activity of the reporter genes. (C) Coimmunoprecipitation. Cell lysates were prepared from HEK293T and SH-SY5Y cells. Coimmunoprecipita- tion was performed with 5 l of -ATXN2 antibody (left) or 1 l of -DDX6 antibody (right). Precipitated proteins were visualized with the antibodies indicated.

Article Snippet: The following antibodies were used: TIA-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), DDX6 (1:500; Novus Biologicals), hemagglutinin (1:500; Roche Diagnostics), FLAG (1:500; Sigma Chemie), DCP1 (1:600; van Dijk et al., 2002), PABP (1:500; (Kuyumcu-Martinez et al., 2004), ATXN2 (1:200; BD Biosciences); MYC (1:600; Upstate Biotechnology, Lake Placid, NY); -goat-Cy3 or -rabbit-Cy3 (1:500; Dianova, Hamburg, Germany), and -mouse-fluorescein isothiocyanate (FITC) (1:500; Dianova).

Techniques: Transformation Assay, Isolation, Membrane, Activity Assay

Journal: Molecular biology of the cell

Article Title: Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

doi: 10.1091/mbc.e06-12-1120

Figure Lengend Snippet: Figure 2. ATXN2 and DDX6 colocalize in stress granules. DU145 cells were treated with 0.5 mM arsenite or heat shocked at 43.5°C for 1 h. Control cells were left untreated at 37°C. Colocalization of endogenous ATXN2 and DDX6 (A), TIA-1 (B), or DCP1 (C). Nuclei were stained with Hoechst. Bars, 10 m.

Article Snippet: The following antibodies were used: TIA-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), DDX6 (1:500; Novus Biologicals), hemagglutinin (1:500; Roche Diagnostics), FLAG (1:500; Sigma Chemie), DCP1 (1:600; van Dijk et al., 2002), PABP (1:500; (Kuyumcu-Martinez et al., 2004), ATXN2 (1:200; BD Biosciences); MYC (1:600; Upstate Biotechnology, Lake Placid, NY); -goat-Cy3 or -rabbit-Cy3 (1:500; Dianova, Hamburg, Germany), and -mouse-fluorescein isothiocyanate (FITC) (1:500; Dianova).

Techniques: Control, Staining

Journal: Molecular biology of the cell

Article Title: Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

doi: 10.1091/mbc.e06-12-1120

Figure Lengend Snippet: Figure 3. ATXN2 overexpression interferes with P- body assembly. (A) SH-SY5Y cells were transiently transfected with plasmids pCMV-MYC-ATXN2-Q22 or pCMV-MYC-ATXN2-Q79. For staining exogenous ATXN2 and endogenous DDX6, cells were incubated with anti- bodies directed against the MYC-tag and DDX6, fol- lowed by treatment with secondary antibodies coupled to FITC or Cy3, respectively. For quantitative analysis, the percentage of P-bodies in nontransfected versus transfected cells was calculated using the AxioVision software. Here, P-bodies of cells were counted in each picture taken and divided through the cell number. The mean value of P-bodies in the cells counted was calculated and SD was weighted. Then, the number of P-bodies counted for the nontransfected cells was set as 100%, and the number of P-bodies counted for the transfected cells was aligned. (B) Twenty-four hours after transfection, SH- SY5Y cells transiently overexpressing MYC-ATXN2(Q22) or MYC-ATXN2(Q79) or HEK293T transiently overex- pressing MYC-ATXN2(Q22) (C) were stained for the MYC-tag and DDX6 or DCP1, respectively. Nuclei were stained with Hoechst. Bars, 10 m. Arrows indicate trans- fected cells.

Article Snippet: The following antibodies were used: TIA-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), DDX6 (1:500; Novus Biologicals), hemagglutinin (1:500; Roche Diagnostics), FLAG (1:500; Sigma Chemie), DCP1 (1:600; van Dijk et al., 2002), PABP (1:500; (Kuyumcu-Martinez et al., 2004), ATXN2 (1:200; BD Biosciences); MYC (1:600; Upstate Biotechnology, Lake Placid, NY); -goat-Cy3 or -rabbit-Cy3 (1:500; Dianova, Hamburg, Germany), and -mouse-fluorescein isothiocyanate (FITC) (1:500; Dianova).

Techniques: Over Expression, Transfection, Staining, Incubation, Software

Journal: Molecular biology of the cell

Article Title: Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

doi: 10.1091/mbc.e06-12-1120

Figure Lengend Snippet: Figure 4. The LSm/LSmAD domain of ATXN2 is ac- countable for the interference with P-body structures. SH-SY5Y cells were transiently transfected with plas- mid pCMV-MYC-ATXN2-LSm/LSmAD or pTL-FLAG- ATXN2-LSm/LSmAD. For staining exogenous ATXN2 and endogenous DDX6, cells were incubated with anti- bodies directed against the MYC- or FLAG-tag and DDX6, followed by treatment with secondary antibodies coupled to FITC or Cy3, respectively. To overexpress an unrelated protein as control, cells were transfected with plasmid pTL-FLAG-endophilin-A3. Nuclei were stained with Hoechst. Bars, 10 m. Arrows indicate transfected cells.

Article Snippet: The following antibodies were used: TIA-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), DDX6 (1:500; Novus Biologicals), hemagglutinin (1:500; Roche Diagnostics), FLAG (1:500; Sigma Chemie), DCP1 (1:600; van Dijk et al., 2002), PABP (1:500; (Kuyumcu-Martinez et al., 2004), ATXN2 (1:200; BD Biosciences); MYC (1:600; Upstate Biotechnology, Lake Placid, NY); -goat-Cy3 or -rabbit-Cy3 (1:500; Dianova, Hamburg, Germany), and -mouse-fluorescein isothiocyanate (FITC) (1:500; Dianova).

Techniques: Transfection, Staining, Incubation, FLAG-tag, Control, Plasmid Preparation

Journal: Molecular biology of the cell

Article Title: Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

doi: 10.1091/mbc.e06-12-1120

Figure Lengend Snippet: Figure 5. Overexpression of ATXN2 does not influ- ence SG formation. DU145 cells were transiently trans- fected with plasmid pCMV-MYC-ATXN2-Q22. Eight hours post transfection, cells were treated with 0.5 mM arsenite for 1 h. Control cells were left untreated at 37°C. For visualization of MYC-tagged ATXN2, cells were incubated with an antibody directed against the MYC- tag and antibodies against DDX6 (A) or TIA-1 (B) under normal and oxidative stress conditions as indicated. Arrows indicate transfected cells. Nuclei were stained with Hoechst. Bars, 10 m.

Article Snippet: The following antibodies were used: TIA-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), DDX6 (1:500; Novus Biologicals), hemagglutinin (1:500; Roche Diagnostics), FLAG (1:500; Sigma Chemie), DCP1 (1:600; van Dijk et al., 2002), PABP (1:500; (Kuyumcu-Martinez et al., 2004), ATXN2 (1:200; BD Biosciences); MYC (1:600; Upstate Biotechnology, Lake Placid, NY); -goat-Cy3 or -rabbit-Cy3 (1:500; Dianova, Hamburg, Germany), and -mouse-fluorescein isothiocyanate (FITC) (1:500; Dianova).

Techniques: Over Expression, Plasmid Preparation, Transfection, Control, Incubation, Staining

Journal: Molecular biology of the cell

Article Title: Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

doi: 10.1091/mbc.e06-12-1120

Figure Lengend Snippet: Figure 7. A reduced ATXN2 level does not seem to affect P-body structures. HEK293T cells were transfected with siControl#1, si- ATXN2#3, or siATXN2#6 molecules or with transfection reagent only (mock). Endogenous levels of ATXN2 and DDX6 were visualized using antibodies against ATXN2 and DDX6. White boxes represent areas represented en- larged in the adjacent picture column. Nuclei were stained with Hoechst. Bars, 10 m.

Article Snippet: The following antibodies were used: TIA-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), DDX6 (1:500; Novus Biologicals), hemagglutinin (1:500; Roche Diagnostics), FLAG (1:500; Sigma Chemie), DCP1 (1:600; van Dijk et al., 2002), PABP (1:500; (Kuyumcu-Martinez et al., 2004), ATXN2 (1:200; BD Biosciences); MYC (1:600; Upstate Biotechnology, Lake Placid, NY); -goat-Cy3 or -rabbit-Cy3 (1:500; Dianova, Hamburg, Germany), and -mouse-fluorescein isothiocyanate (FITC) (1:500; Dianova).

Techniques: Transfection, Staining

Journal: Fish Physiology and Biochemistry

Article Title: Thymol and menthol as anaesthetics for short transportation of zebrafish larva

doi: 10.1007/s10695-025-01530-x

Figure Lengend Snippet: a Nrf2 protein levels in 96 hpf zebrafish larvae following 4 h transportation. b Representative Western blots of Nrf2. Data are expressed as mean ± SD for parametric data distribution. Statistical analysis was performed using Student’s t test (unpaired) to compare the naïve group with the control group, and used one-way ANOVA followed by Dunnett’s multiple-comparison test for comparation of the control and anaesthetized groups. Asterisks indicate significative differences between the control and anaesthetized groups ( p < 0.05)

Article Snippet: The primary antibodies for GAPDH (1:1000, GTX100118, 36 kDa, GeneTex) and Nrf2 (1:20, PCRP-NFE2L2–1D12, 68 kDa, DSHB) were used overnight following the blockade of the membrane with 5% BSA.

Techniques: Western Blot, Control, Comparison