Journal: Journal of cachexia, sarcopenia and muscle

Article Title: The Innovative Role of Nuclear Receptor Interaction Protein in Orchestrating Invadosome Formation for Myoblast Fusion.

doi: 10.1002/jcsm.13598

Figure Lengend Snippet: FIGURE 1 | NRIP is an actin-binding protein. (A) NRIP directly interacted with actin in vitro. Upper panel: His-MBP and His-MBP-NRIP proteins were examined using Coomassie blue staining. The arrows indicate His-MBP and His-MBP-NRIP. Lower panel: GST and GST-actin proteins indicated by arrows. (B) Left panel: His pull-down assy. Right panel: GST pull-down assay. The pull-downed extracts were analysed by western blotting with an anti-His antibody to detect His-MBP and His-MBP-NRIP and an anti-GST antibody to detect GST-actin. (C) NRIP interacted with actin in C2C12 myotubes. The protein extracts from C2C12 myotubes were immunoprecipitated with anti-NRIP or anti-alpha-actin and immunoblotted with indicated antibodies. The rabbit normal IgG (rIgG) served as the antibody control for anti-NRIP, while the mouse normal IgG (mIgG) was the control for anti-alpha-actin. (D) NRIP interacted with actin in cells. Cell extracts (1 mg) from 293T cells co-transfected with Flag-NRIP and mCherry-actin were immunoprecipitated with anti-mCherry (actin) (left panel) or with anti-Flag (NRIP) (right panel) and immunoblotted with indicated antibodies. (E) Subcellular localization of NRIP in C2C12 cells. Nuclear (N), cytosolic (C), and membrane (M) fractions from C2C12 cells were extracted as described in Methods. EGFR was a positive control of membrane proteins, PCNA was a positive control for nuclear proteins, and GAPDH was a positive control for cytosolic proteins. % of total NRIP was determined as the intensity ratio of nuclear, cytosolic, or membrane NRIP to total NRIP (N = 3). (F) Endogenous NRIP was located at the plasma membrane, nucleus and cytoplasm. C2C12 myoblasts were differentiated for 4 days and stained with anti-NRIP (green), phalloidin for the cell membrane (red, F-actin staining), and DAPI for the nucleus (blue). Arrowheads: either NRIP or F-actin at the plasma membrane. Arrows: NRIP in the cytoplasm. Asterisks: NRIP in the nucleus. Scale bar: 20 μm.

Article Snippet: Protein lysates (50 μg) were subjected to western blotting with indicated primary antibodies: anti- NRIP (A302- 434A, Novus, 1:2000), anti- EGF receptor (#2646, Cell Signalling, 1:1000), anti- DsRed (632496, TaKaRa, 1:10 000), anti- actin (ab179467, Abcam, 1:10 000), anti- EGFP (ab6556, Abcam, 1:10 000), anti- flag (ab1162, Abcam, 1:10 000), anti- MyHC (ab124205, Abcam, 1:2000), anti- Tks5 (sc- 376211, Santa Cruz, 1:1000), anti- cortactin (sc55579, Santa Cruz, 1:1000), anti- His (66005- 1- Ig, Proteintech, 1:10 000), anti- GST (sc- 459, Santa Cruz, 1:10 000), anti- PCNA (#2586, Cell Signalling, 1:10 000), and anti- GAPDH (LFPA0212, AbFrontier, 1:10 000).

Techniques: Binding Assay, In Vitro, Staining, Pull Down Assay, Western Blot, Immunoprecipitation, Control, Transfection, Membrane, Positive Control, Clinical Proteomics

Journal: Developmental biology

Article Title: The Transition from Proliferation to Differentiation Is Delayed in Satellite Cells from Mice Lacking MyoD

doi: 10.1006/dbio.1999.9284

Figure Lengend Snippet: Micrographs of myofiber cultures from control wildtype mice (A, B, and C) and MyoD−/− mice (D and E) reacted via double immunofluorescence with monoclonal antibodies against the nuclear antigen PCNA, MyoD, or myogenin along with the polyclonal antibody against ERK1/ERK2. All micrographs depict cultures that were maintained in basal medium + FGF2 at 2 ng/ml. (A and A′) A day 4 culture reacted with anti-PCNA and anti-ERK. (B and B′) A day 3 culture reacted with anti-MyoD and anti-ERK. (C and C′) A day 4 culture reacted with anti-myogenin and anti-ERK. (D and D′) A day 3 culture reacted with anti-PCNA and anti-ERK. (E and E′) A day 5 culture reacted with anti-myogenin and anti-ERK. (A″, B″, C″, D″, and E″) Parallel micrographs of DAPI stain which highlight all myofiber and satellite cell nuclei. For each antibody combination, reactivity with the monoclonal antibody was visualized with a fluorescein-labeled secondary antibody and reactivity with the anti-ERK antibody was visualized rhodamine-labeled secondary antibody. Arrows in parallel images point to the same locations of cells as identified by immunostaining and by DAPI. Note that not all positive nuclei or cells on the myofibers are in the same focal plane. Bar, 34 μm.

Article Snippet: A mouse monoclonal antibody against PCNA was from Boehringer Mannheim (Indianapolis, IN).

Techniques: Control, Immunofluorescence, Bioprocessing, Staining, Labeling, Immunostaining

Journal: Developmental biology

Article Title: The Transition from Proliferation to Differentiation Is Delayed in Satellite Cells from Mice Lacking MyoD

doi: 10.1006/dbio.1999.9284

Figure Lengend Snippet: Temporal appearance of cells or nuclei positive for ERK1/ERK2 and PCNA (A), MyoD (B), and myogenin (C) in cultures of myofibers isolated from Balb/C mice. Cultures were maintained in basal medium ± FGF2 at 2 ng/ml and the medium was changed daily. Plates were collected every 24 h and reacted via double immunofluorescence with the polyclonal antibody against ERK1/ERK2 in combination with the monoclonal antibodies against PCNA, MyoD, and myogenin. Immunostaining was performed as detailed in the legend to Fig. 1. Two parallel 35-mm plates were analyzed for each time point. For each panel, the total number of ERK+ cells and the number of doubly positive cells (PCNA+/ERK+, MyoD+/ERK+, myogenin+/ERK+) were determined. Nuclei positive for PCNA, MyoD, or myogenin which were not within ERK+ cells were never detected. The total number of ERK+ cells, shown in A, was similar for all three panels. Cells were scored as the number of positives on each individual fiber, analyzing 30 fibers per plate. Total positives were then averaged for duplicate plates and this value was eventually expressed per 10 fibers as indicated on the y axis. The error bar depicts the range of the variation between the duplicate plates of individual experiments.

Article Snippet: A mouse monoclonal antibody against PCNA was from Boehringer Mannheim (Indianapolis, IN).

Techniques: Isolation, Immunofluorescence, Bioprocessing, Immunostaining

Journal: Developmental biology

Article Title: The Transition from Proliferation to Differentiation Is Delayed in Satellite Cells from Mice Lacking MyoD

doi: 10.1006/dbio.1999.9284

Figure Lengend Snippet: Temporal appearance of cells or nuclei positive for ERK1/ERK2 and PCNA (A) and ERK1/ERK2 and myogenin (B) in cultures of myofibers isolated from MyoD−/− mice. Cultures were maintained in basal medium (±FGF2 at 2 ng/ml) and the medium was changed daily. Plates were collected every 24 h and reacted via double immunofluorescence with the polyclonal antibody against ERK1/ERK2 in combination with the monoclonal antibodies against PCNA and myogenin. Immunostaining was performed as detailed in the legend to Fig. 1. Two parallel 35-mm plates were analyzed for each time point. For each panel, the total number of ERK+ cells and the number of doubly positive cells (PCNA+/ERK+ or myogenin+/ERK+) were determined. Nuclei positive for PCNA or myogenin which were not within ERK+ cells were never detected. Also, immunostaining with the antibody against MyoD could not detect immunostained nuclei or cells. Cells were scored as the number of positives on each individual fiber, analyzing 30 fibers per plate. Total positives were then averaged for duplicate plates and this value was eventually expressed per 10 fibers as indicated on the y axis. The error bar depicts the range of the variation between the duplicate plates of individual experiments.

Article Snippet: A mouse monoclonal antibody against PCNA was from Boehringer Mannheim (Indianapolis, IN).

Techniques: Isolation, Immunofluorescence, Bioprocessing, Immunostaining