Actinin

IF staining experiments were performed utilizing either HEK293 cells or freshly isolated rat ventricular myocytes, as previously described (15 (link)) using the following primary and secondary antibodies: PKP4 (ThermoFisher Scientific, PA5-66855, dilution 1/500), Kir2.1 (Alomone Labs, Jerusalem, Israel, AGP-044, dilution 1/20), Actinin (Sigma, SAB4503474, 1/300), GM130 (Novus Biologicals, NBP2-53420; dilution 1/200), FLAG (Sigma-Aldrich; F3165, dilution: 1/50), Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-guinea pig and Alexa Fluor 647 donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA, dilution 1/400).

Hearts were homogenized using homogenization buffer containing 1 × Hsaio TBS, PMSF (1:1000), 1:1000 Protease Inhibitor cocktail set 3 (EMD Millipore #539134), 1:1000 PhosphotaseArrest II (GBioscience #786-451), and 1:1000 PhosphotaseArrest III (GBioscience #786-452). Samples were centrifuged at 10,000 rpm and supernatant was isolated. Protein supernatant concentration was determined using BCA (Thermo #23225). Samples were incubated with anti-V5 antibody (ABCAm #ab184144) at 1:1000 overnight at 4 °C. G-protein beads were then incubated with samples for 4 hours. Samples were centrifuged at 3000 rpm and beads were isolated. Beads were washed twice and centrifuged at 3000 rpm. Beads were centrifuged at 10,000 rpm and supernatant was entirely removed. Beads were then resuspended in 2× Laemmli Buffer with beta-mercaptoethanol. Samples were run on 10% Tris-Glycine gels (BioRad #5671034), and transferred to PVDF membranes (EMD Millipore #IPVH00010). Primary antibodies used were Anti-V5 antibody (ABCAm #ab184144) at 1:1000 and Actinin (Sigma-Aldrich #A2066) at 1:1000. Protein signals were revealed by ECL (Pierce #32106) using a GE Amersham Imager 600UV system. Image analysis was performed using ImageJ. Bands of interest were normalized to a loading control. Statistical analysis of the bands was performed using Student’s t-test in GraphPad Prism.

Cardiomyocytes were washed twice with cold 1X PBS and lysed with RIPA buffer (Sigma) containing 1X mammalian protease inhibitor (Sigma, P8340). Proteins in cell homogenates were separated on SDS-PAGE and transferred to nitrocellulose membrane (LI-COR). Soluble and insoluble protein fractions were prepared as reported before^{68 (link)}. Antibodies against Troponin I (Millipore), Nef (NIH), LC3-II (Sigma), Rab7 (Cell Signaling), p62 (Cell signaling), Beclin 1 (Cell Signaling, 3495) Akt (Cell Signaling, 9272) and pAkt (Cell Signaling), GFP (Clontech), Actinin (Sigma) were used for the Western blot analysis.

Apoptosis was detected in heart sections and ARCMs with triple staining: DAPI, TUNEL (In Situ Cell Death Detection Kit, Roche), and actinin (A7811, Sigma). TUNEL was performed following the manufacturer’s instructions, followed by overnight incubation with primary α-actinin antibody at 4° C. Next, α-actinin signals were obtained by incubation with donkey anti-mouse-Alexa-594 conjugate (715-585-150, Jackson ImmunoResearch) for 2 hr at room temperature. Finally, the sections were mounted with DAPI-containing Vectashield (H-1200, Vector Laboratories). Fluorescent images were acquired with an Olympus BX51 upright microscope and a Coolsnap EZ camera (Photometrics). Specific band pass filter sets for DAPI, FITC and Texas red were used. For quantification, ten fields were acquired per sample, and images were analyzed using ImageJ.

The proband underwent a muscle biopsy after giving written informed consent, according to Institutional guidelines. Morphological examination was performed according to standard procedures.
Immunohistochemical (IHC) analyses were performed using monoclonal antibodies directed against three different epitopes of dystrophin (rod-domain diluted 1:10, NH₂-domain diluted 1:10, COOH-domain undiluted, all from Novocastra, Newcastle upon Tyne, UK), sarcoglycans (alpha-sarcoglycan 1:20, gamma-sarcoglycan 1:10, Novocastra, Newcastle upon Tyne, UK), and caveolin-3 (1:1000, BD Transduction Laboratories, Franklin Lakes, NJ, USA) as previously described [23 (link)].
Forty micrograms of muscle protein lysates was probed for calpain-3 (Novocastra, 2C4, 1:100), dysferlin (Novocastra, Newcastle upon Tyne, UK, Hamplet-2 1:1000), dystrophin (rod-domain 1:200 and COOH-term 1:80), actinin (Sigma, St. Louis, MO, USA, 1:10,000), and sarcoglycans (alpha-sarcoglycan 1:280, beta-sarcoglycan 1:65, gamma-sarcoglycan 1:200, delta-sarcoglycan 1:60, all from Novocastra) expressions by Western blot (WB) analysis on a 10% polyacrylamide gel.

Mice were administered 0.35 mg/L 5-ethynyl-2'-deoxyuridine (EdU) for 28 days via drinking water, changed every third day. Mice were then administered pentobarbital IP, heart removed and placed in PBS. The aorta was cannulated, and the heart perfused with PBS for 5 min followed by perfusion at ~100 mmHg with 4% paraformaldehyde for approximately 10 min or until flow rate was greatly reduced. Hearts were stored in fresh 4% formaldehyde for 24 hr at 4 degrees. Hearts were washed with phosphate buffer and imbedded in 4% low melting point agarose. Sequential transverse sections from the heart of WT and TG^AC8 transgenic mice were sectioned on the Leica Vibratome VT1000s from 200 u-300uM. Sections were permeabilized with 0.2% triton, glycine, and 2% DMSO for 3 days. EDU labeling (Click Chemistry Tools), and primary antibodies Vimentin 1:500 (Synaptic Systems), Actinin (Sigma) 1:500, WGA 1:500 (Vector Labs), 4′,6-diamidino-2-phenylindole DAPI 1:300 (Sigma). Five microscopic fields, per mouse, in the left ventricle were visualized in cardiomyocytes via fluorescent imaging (Zeiss LSM 980) at ×400 magnification, and the number of cardiac nuclei staining positively for EdU was counted in each.