Journal: Molecular and Cellular Biology

Article Title: Defective Flux of Thrombospondin-4 through the Secretory Pathway Impairs Cardiomyocyte Membrane Stability and Causes Cardiomyopathy

doi: 10.1128/MCB.00114-18

Figure Lengend Snippet: Impaired sarcolemmal glycoprotein trafficking in hearts overexpressing secretion-defective Thbs4. (A) Western blotting for membrane glycoproteins purified from hearts of transgenic mice overexpressing wild-type Thbs4 (DTG-Thbs4) or the secretion-defective mutant (DTG-mCa2+). Cadherin was used as a loading control. (B) Representative experiment of immunocytochemistry for β-sarcoglycan and the Golgi marker GM130 in cardiomyocytes isolated from transgenic hearts of the indicated genotype. Scale bar = 50 μm. Similar results were obtained in 2 additional experiments.

Article Snippet: Briefly, paraformaldehyde-fixed paraffin-embedded cardiac sections were incubated in blocking solution (PBS, 5% goat serum, 1% bovine serum albumin [BSA], 1% glycine, 0.2% Triton X-100) for 1 h and then with anti-Thbs4 primary antibody (Santa Cruz) diluted 1:500 in blocking solution overnight, followed by incubation with Alexa Fluor-labeled secondary antibody (Invitrogen; 1:1,000) for 1 h. For immunocytochemistry, adult mouse cardiomyocytes were isolated from mouse hearts by Langendorff perfusion, fixed in 4% paraformaldehyde, and immunostained in suspension as described previously ( 51 ) using antibodies against β-sarcoglycan (Novus Biologicals) and GM130 (BD Biosciences) coupled to Alexa Fluor-labeled secondary antibodies (Invitrogen).

Techniques: Western Blot, Membrane, Purification, Transgenic Assay, Mutagenesis, Control, Immunocytochemistry, Marker, Isolation