Journal: The Journal of Neuroscience
Article Title: Sarcoglycan Alpha Mitigates Neuromuscular Junction Decline in Aged Mice by Stabilizing LRP4
Figure Lengend Snippet: Increasing LRP4 protein stability by SGα. A , LRP4 interaction with SGα, but not SGδ. Flag-LRP4 was precipitated from HEK293 cells cotransfected with SGα-V5 or SGδ-V5 and probed with anti-V5 antibody. Lysates were probed with the same antibodies as control. B , In vivo interaction between LRP4 and SGα. Homogenates were prepared from muscles of Flag-Lrp4 transgenic mice and subjected to IP with anti-SGα antibody. Immunocomplex was probed with anti-Flag and SGα antibodies. C , mRNA expression of sarcoglycan complex molecules in diaphragm synaptic region; t (4) = 9.97, ** p = 0.0026 for Sgca ; t (4) = 8.87, ** p = 0.0039 for Sgcb ; t (4) = 10.8, *** p = 0.0004 for Sgcg ; N = 3 mice per group, unpaired t test. D , Reduced SGα protein level in aged mice. Top, Representative blots. Bottom, Quantitative data; t (4) = 5.66, * p = 0.015; N = 3 mice per group, unpaired t test. E , Discontinuous staining of SGα in aged TA muscles. White arrowhead, NMJs identified by α-BTX with discontinuous SGα staining. Scale bar, 20 μm. F , G , Increased LRP4 stability in HEK293 cells expressing SGα. HEK293 cells were transfected with Flag-LRP4 without (control) or with SGα-V5 and incubated with CHX. Flag-LRP4 levels were analyzed at indicated times. F , Representative blot; G , quantitative data; F (1,16) = 56.5, ** p = 0.0021 and 0.0095, *** p = 0.0001; N = 3, two-way ANOVA.
Article Snippet: Antibodies used were as follows: AChRδ (88B; 1:2000 for Western blot) from ThermoFisher Scientific; AChRε (ab65180; 1:2000 for Western blot), SGα (ab189254; 1:500 for staining and 1:2000 for Western blot) from Abcam; Ubiquitin (sc-8017; 1:1000 for Western blot), AChRα (sc-65829; 1:1000 for Western blot), and AChRβ (sc-11371; 1:1000 for Western blot) from Santa Cruz Biotechnology; DOK7 (AF6398; 1:1000 for Western blot) from R & D Systems; neurofilament (C28E10; 1:500 for staining) and synapsin (D12G5, 1:500 for staining) from Cell Signaling Technology; GAPDH (NB 600-501; 1:3000 for Western blot) from Novus; V5 (V8012; 1:1000 for Western blot), laminin (041M4799; 1:200 for staining) and GFP (11814460001; 1:3000 for Western blot and 1:500 for staining) from Sigma Aldrich.
Techniques: In Vivo, Transgenic Assay, Mouse Assay, Expressing, Staining, Transfection, Incubation