Article Title: Thrombospondin expression in myofibers stabilizes muscle membranes
Figure Lengend Snippet: Thbs4 enhances stabilizing proteins at the sarcolemma and directly interacts with integrins. ( A , B ) Representative Western blots of sarcolemmal protein extracts from the quadriceps of the indicated groups of mice at three months of age (n = 4–5 biological replicates). Sgcd -/- Tg and mdx Tg indicate Sgcd -/- and mdx with skeletal muscle specific Thbs4 overexpression, respectively. Ponceau staining of a nonspecific band and dihydropyridine receptor α1 (Cav1.1) were used as loading controls. Abbreviations: Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; α-DG, α-dystroglycan; β-DG, β-dystroglycan; δ-SCG, δ-sarcoglycan; α-SCG, α-sarcoglycan; β-SGC, β-sarcoglycan; β1D-, α7- and α5-itg (integrin). ( C ) Immunoblots for β1D- and α7-Integrin (Itg), β-dystroglycan (DG) and Thbs4 (Flag) from neonatal rat ventricular myocyte extracts immunoprecipitated with a Flag antibody (Thbs4). Adβgal was used as a control infection (n = 3 biological replicates). An adenovirus expressing a Flag-tagged Thbs4 protein was used to achieve high level of this protein to identify the interaction. ( D , E ) Representative Western blots for Thbs4, α5- and β1D-integrin (itg) from intracellular vesicular isolates from WT and Thbs4 Tg quadriceps ( D ) or WT and Sgcd -/- quadriceps ( E ) that were immunoprecipitated with an antibody raised against the cytoplasmic domain of β1D-integrin (n = 3 biological replicates), showing that Thbs4 and α5-integrin localize to β1D-integrin-positive intracellular vesicles. α-tubulin and Gapdh are presented as loading control. DOI: http://dx.doi.org/10.7554/eLife.17589.019
Article Snippet: Other primary antibodies used in this study included: α-actinin (sarcomeric, A7811; 1/1000); Armet (Abcam, ab67271; 1:1000); BiP (Cell Signaling Technology, Danvers, MA, 3177; 1:1000); calreticulin (Cell Signaling Technology, 2891; 1:1000); dihydropyridine receptor α1 (Cav1.1; Thermo Fisher Scientific, MA3-920; 1:1000); dysferlin (Abcam, ab124684 [JAI-1-49-3]; 1:1000); CLIC3(Santa Cruz, sc-390006; 1:200); α-dystroglycan(EMD Millipore, 05–593; 1:500); β-dystroglycan (Development Studies Hybridoma Bank, MANDAG2 clone 7D11; 1:100); dystrophin (Sigma-Aldrich, D8043; 1:1000); gadph (Fitzgerald, Acton, MA, 10R-G109A; 1:10000); GFP (Abcam, Ab290; 1:1000); Flag (Cell Signaling Technology, 2368; 1:1000); α5-integrin (EMD Millipore, Billerica, MA, AB1928; 1/1000); α7-integrin (Abacm, ab203254; 1:1000); β1D-integrin (Millipore, MAB1900, 1:500); Laminin (Abcam, ab11575; 1/1000); Pdi (Cell Signaling Technology, 2446; 1:1000); rab3 (Abcam, ab3336; 1:1000); rab4 (Cell Signaling Technology, 2167; 1:1000); rab5 (Sigma-Aldrich, R7904; 1:500); rab6 (Cell Signaling Technology, 4879; 1:1000); rab7 (Cell Signaling Technology, 9367; 1:1000); rab8 (Abcam, ab188574; 1:1000); rab11 (Abcam, ab95375; 1:1000); rab24 (BD Biosciences, Franklin Lakes, NJ, 612174; 1:1000); Sar1 (Abcam, ab125871; 1:1000); α-sarcoglycan (Development Studies Hybridoma Bank, IVD3(1)A9; 1:100); β-sarcoglycan (Novus Biologicals, Littleton, CO, NBP1-90300; 1:1000); δ-sarcoglycan (Abcam, ab137101; 1:500); sarcospan (Santa Cruz Biotechnology, sc-393187; 1:200); α-tubulin (Santa Cruz Biotechnology, sc-8035; 1:1000 dilution); utrophin (Development Studies Hybridoma Bank, MANCHO3(8A4); 1:50) and Wash1 (Abcam, ab157592; 1:1000).
Techniques: Western Blot, Mouse Assay, Over Expression, Staining, Immunoprecipitation, Infection, Expressing