Journal: Differentiation; research in biological diversity

Article Title: The combination of glycosaminoglycans and fibrous proteins improves cell proliferation and early differentiation of bovine primary skeletal muscle cells.

doi: 10.1016/j.diff.2013.06.006

Figure Lengend Snippet: Fig. 2. Localization and expression of myogenic regulatory transcription factors (MRFs) during proliferation and differentiation. (A) Proliferating cells were fixed with 2% PFA, immunostained with Alexa 488-Phalloidin (green), and rabbit anti-MyoD, followed by DyLight 649-conjugated donkey anti-rabbit (red) before fluorescence microscopy analysis. Nuclei were stained with DAPI (blue). Scale bar 10 mM. (B) Cells differentiated for 3 days were fixed with 2% PFA, immunostained with Alexa 488-Phalloidin (green), and goat anti-Myogenin, followed by DyLight 649-conjugated donkey anti-goat (red) before fluorescence microscopy analysis. Nuclei were stained with DAPI (blue). Arrows indicate Myogenin positive cells. Scale bar 10 mM. (C) Differentiating cells were fixed with 2% PFA, immunostained with Alexa 488-Phalloidin (green) and rabbit anti-Desmin, followed by DyLight 549-conjugated mouse anti-rabbit (red) before fluorescence microscopy analysis. Nuclei were stained with DAPI (blue). Scale bar 10 mM. (D) Left: a representative western blot showing the expression level of MyoD and Desmin after 0 and 3 days of differentiation. Cell lysates were subjected to western blotting using antibodies to MyoD, Desmin, and Tubulin (loading control). Right: quantification of Western blots. Bars show the expression of Desmin and MyoD, relative to tubulin, after 0 and 3 days of differentiation. The graph represents the average of three experiments with SD.

Article Snippet: Alexa 488-conjugated goat anti-mouse and DyLight 549- conjugated mouse anti-rabbit were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Techniques: Expressing, Microscopy, Staining, Western Blot, Control

Journal: Differentiation; research in biological diversity

Article Title: The combination of glycosaminoglycans and fibrous proteins improves cell proliferation and early differentiation of bovine primary skeletal muscle cells.

doi: 10.1016/j.diff.2013.06.006

Figure Lengend Snippet: Fig. 6. Desmin staining of myotubes on different surface coatings after 1, 3 or 5 days in differentiation medium. Differentiating cells were fixed with 2% PFA and immunostained with rabbit anti-Desmin, followed by DyLight 549-conjugated mouse anti-rabbit (yellow) before fluorescence microscopy analysis. Nuclei were stained with DAPI (blue). Scale bar 20 mM.

Article Snippet: Alexa 488-conjugated goat anti-mouse and DyLight 549- conjugated mouse anti-rabbit were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Techniques: Staining, Microscopy

Journal: Scientific Reports

Article Title: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

doi: 10.1038/s41598-017-06319-4

Figure Lengend Snippet: Co-immunoprecipitation (IP) of the Nef-ATG8 complex. Co-IP studies were carried out with lysates prepared from HEK293 cells stably expressing Nef-DsRed ( A–C ) and cotransfected with plasmids encoding for the indicated YFP-ATG8 constructs ( D–F ). ( A ) and ( D ) show samples before immunoprecipitation. In ( A ) an anti-GABARAPL2 (a), an anti-GABARAP (b), an anti-GABARAPL1 (c), and an anti-LC3B antibody (d) were used to detect the different ATG8s. ( B ) Immunoprecipitates with anti-DsRed antibody followed by SDS-PAGE and immunoblotting with various ATG8 antibodies (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B). ( C ) In a reciprocal set of experiments, immunoprecipitates were carried out with various ATG8 (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B) antibodies followed by SDS-PAGE and immunoblotting with an anti-DsRed antibody. In ( D ) an anti-DsRed antibody was used to detect Nef fused to DsRed and an anti-YFP antibody to detect YFP and the ATG8s fused to YFP. ( E ) Immunoprecipitates with an anti-DsRed antibody or ( F ) with an anti-GFP antibody followed by SDS-PAGE and immunoblotting with the antibodies are indicated. Data are representative of three independent experiments (U: unbound material; E: eluate fraction). Full-length western blots are presented in Supplementary Figs and .

Article Snippet: Rabbit polyclonal antibodies raised against GABARAP (18723-1-AP), GABARAPL2 (18724-1-AP), GABARAPL1 (18721- 1-AP), and LC3B (18725-1-AP) were obtained from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Construct, SDS Page, Western Blot

Journal: Scientific Reports

Article Title: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

doi: 10.1038/s41598-017-06319-4

Figure Lengend Snippet: GABARAP residues S53 and S62 are essential for Nef binding. ( A ) Human ATG8s sequence alignment indicates putative key residues for Nef binding specificity. Protein names of ATG8s showing Nef binding during pull-down and immunoprecipitation analysis are given in different shades of yellow, and include all members of the GABARAP subfamily. Protein names of LC3B and of the other LC3s are given in different shades of brown. Purple arrows indicate residues of GABARAP showing chemical shift changes higher than 0.05 ppm (see Fig. ) upon Nef titration. Residues forming HP1 and HP2 are shaded in light and dark blue, respectively. Red asterisks highlight putative key positions for determining Nef-binding specificity. The corresponding amino acids found in GABARAP and LC3B at these positions are highlighted in red. ( B ) Visualization of HP1 and HP2 on the surface of the GABARAP structure [PDB ID: 1KOT]. ( C ) Structural overlay of GABARAP and LC3B with the putative key residues necessary for Nef-binding highlighted. Cartoon representations of GABARAP (yellow) and LC3B (brown) [PDB ID: 1V49] showing their regular secondary structure elements demonstrate that S53 and F62 of GABARAP as well as the corresponding D56 and K65 of LC3B are surface exposed, and therefore are available for ligand binding. Details highlight the side chain orientations of the key residues, for this the structures have been rotated appropriately. ( D ) Nef-conjugated or free Sepharose beads (control) were incubated with GABARAP(S53D/F62K) or LC3B(D56S/K65F) mutants. The input, the unbound material of the flow through (U),the wash (W) fractions and the eluate (E) fractions were subjected to SDS-PAGE and visualized by CBB staining. Full-length gels are presented in Supplementary Fig. .

Article Snippet: Rabbit polyclonal antibodies raised against GABARAP (18723-1-AP), GABARAPL2 (18724-1-AP), GABARAPL1 (18721- 1-AP), and LC3B (18725-1-AP) were obtained from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Titration, Ligand Binding Assay, Control, Incubation, SDS Page, Staining