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