B) Detection with the polyclonal antibody described by Peters et al
B) Detection with the polyclonal antibody described by Peters et al. TAK-071 the dystrophin associated glycoprotein complex in skeletal muscle mass and has a prominent role in muscle mass and neuromuscular development (1). SNTA is also involved in cardiac pathologies like long-QT syndrome and sudden infant death syndrome (1). Altered expression of SNTA in esophageal, belly, lung, colon, rectal, and breast cancerous tissues suggests a function in carcinogenesis (1). SNTA has been further shown to stabilize ABCA1 which is a central regulator of lipid metabolism (7). ABCA1 and SNTA are expressed in the liver (7; 8) but hepatic ABCA1 is not reduced in mice lacking SNTA and beta 2 syntrophin (SNTB2) (9). To find out whether SNTA has any role in the liver we intend to study hepatic TAK-071 SNTA expression in diseased liver tissues by immunoblot analysis. To test for the specificity of commercially available SNTA antibodies liver tissue of C57BL/6 mice and SNTA deficient mice (10) was used. Liver tissue was solubilized in radioimmunoprecipitation assay lysis buffer (50 mM Tris HCl, pH 7.5, 150 mM NaCl, 1% v/v Nonidet P-40, 0.5% v/v sodium desoxycholate and 0.1% v/v SDS). Protein (20 g) was separated by SDS-polyacrylamide gel electrophoresis (15 % acrylamide) TAK-071 and transferred to PVDF TAK-071 membranes (Bio-Rad, Munich, Germany). Incubations with the primary antibodies were performed in 1.5% BSA in TBS, 0.1% Tween at 4C overnight. Secondary antibodies were from Dianova (Hamburg, Germany) and were diluted 1:5000 fold for anti-rabbit and anti-mouse immunoglobulins and 1:1000 fold to detect goat antibodies. Incubations were performed in 5% low-fat milk powder in TBS, 0.1% Tween at RT for 1 h. Detection of the immune complexes was carried out with the ECL Western blot detection system (Amersham Pharmacia, Deisenhofen, Germany). For the generation of SNTA deficient mice the first exon and part of the promoter had been removed and it is very unlikely that any truncated syntrophin is usually expressed in these knock-out animals (10). The recently explained SNTA antibody (11) recognizes a peptide sequence (RQPSSPGPQPRNLSEA) in the PH1b domain name and was raised in rabbits. This antibody (1:1000 fold diluted) does not generate a band in the liver of SNTA?/? mice (Fig. 1A, B). The polyclonal SNTA antibody from Abcam (ab11187; Cambridge, UK) was raised in rabbits using the peptide explained above (amino TAK-071 acids 191-206 of mouse SNTA) as immunogen. The antibody was tested at a dilution of 1 1:2000 as suggested by the company. This antibody detects a protein of about 60 kDa in the liver of wild type but not SNTA?/? animals (Fig. 1A). A SNTA antibody raised in goat was ordered from Thermo Fisher Scientific Pierce (PA1-9107). The immunogen was a synthetic peptide corresponding to the N terminal amino acids ASGRRAPRTGLLE of SNTA. For immunoblot analysis 1.5 g/ml antibody was used. A band of about 60 kDA was detected in PRKMK6 the liver of wild type and SNTA?/? mice (Fig. 1A). Next monoclonal antibodies were tested. Monoclonal antibodies were purchased from Sigma-Aldrich (SAB4200213) and Life Span BioScience (LS-C89921). The immunogens used were Torpedo electric organ membranes and whole purified syntrophin from Torpedo californica electric organ postsynaptic membrane, respectively (data linens provided by the companies). Both antibodies were used at a concentration of 1 1 g/ml. A band of about 60 kDA was detected in.