Mouse anti α actin

For protein extraction, 50 μl of laemmli buffer (0.05% (w/v) bromophenol blue, glycerol (30% (v/v)), 5 M EDTA, NaOH solution, 6% (w/v) SDS and 1.875 M Tris pH 8.8 solution) and 1% (v/v) Dithiothreitol, (all from Sigma-Aldrich, USA) were used. Samples were macerated and denatured for 20 min at 45 °C followed by 20 min 65 °C and finally 10 min at 90 °C. For the immunoblotting assay, 10 μg of total protein extract were resolved in a 12% SDS gel and transferred to a nitrocellulose membrane for 10 min in Trans-Blot Turbo transfer system (Bio-Rad, USA). Membranes were blocked in Tris-buffered saline (TBS) with 0.1% tween 20 (TBS-T) containing 5% BSA and afterwards incubated overnight at 4 °C, with the polyclonal primary antibodies in 1% BSA: rabbit anti-LC3A/B Antibody (1:1000) (Cell-Signaling Technology, USA), mouse anti‐p62 (1:1000) (Abcam, UK), rabbit anti-human caspase 3 (1:700) (Cell Signaling Technology, USA) and mouse anti‐alpha‐actin (Millipore, USA). Secondary antibodies (HRP, anti‐rabbit, anti‐mouse) were from Bio‐Rad, USA (1:5000). Blots were treated with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, USA) or Clarity Western ECL Substrate (Bio‐Rad, USA). Digital images and densitometry analysis of the bands were obtained in a ChemiDoc XRS System (Bio‐Rad, USA) with Quantity One software V4.6.5 (Bio‐Rad, USA), respectively.

For western blotting, the brain and ventral nerve chord of larval fillets were removed and subjected to different stimulation conditions. Afterwards, the fillets were crushed in 1xSDS sample buffer and boiled for 5 min. Dilutions for primary antibodies were as follows: mouse anti-α-actin, 1:20000 (Sigma); chicken anti-GFP, 1:5000 (Invitrogen).

Cells at different time points of differentiation were fixed using 4% paraformaldehyde (PFA). Samples were then subjected to treatment with NH₄Cl and blocking with 0.3% TritonX-containing BSA before incubation with the primary antibodies. Mouse anti SSEA-1 (Santa Cruz Biotechnology, USA), 1:200, over-night at 4 °C; rabbit anti-PKD1 (Santa Cruz, USA), 1:200, 2 h at room temperature (RT); rabbit anti-PKD2 (Orbigen, USA), 1:1000, 1 h at RT; mouse anti-Oct3/4 (Santa Cruz, USA), 1:200, overnight at 4 °C; rat anti-CD31, (Becton Dickinson, USA), 1:100, 1 h RT; mouse anti α-Actin (Sigma Aldrich, Germany) 1:100, 1 h 37 °C. Samples were further incubated with fluorescence labelled secondary antibodies Alexa Fluor® 488 (green), Alexa Fluor® 568 (red), Alexa Fluor® 647 (magenta) (Life-technologies, all diluted 1:500). For germ layer-specific staining we used the chicken anti β–tubulin-III antibody ((TUBB3), Millipore, Billerica, MA), 1:1000, goat anti-human Brachyury (R&D Systems, Minneapolis, MN, USA, www.rndsystems.com), 1:100, o.N. 4°, AF2085 and goat anti-human SOX17 (R&D Systems), 1:500, o.N. 4°, AF1924. Nuclei were stained with DAPI (blue) (1:20,000). Images were captured using an upright fluorescence Zeiss Axioimager Z1 microscope and analysed using Axiovision software (Zeiss, Germany).

The isolated ventricular myocytes were lysed in lysis buffer containing (in mM) 150 NaCl, 20 HEPES, 1 EDTA, and 0.1 PMSF, as well as 1% Triton X-100 (in μg ml⁻¹), at pH 7.2, and the lysates were centrifuged at 13,000 g for 10 min. The proteins were then immunoprecipitated with rabbit IgG (Sigma), mouse anti-SERCA (Affinity BioReagents), mouse anti-α-actin (Sigma) or mouse anti-PLB antibodies (Affinity BioReagents) (1:100 dilution). The immune complexes were subsequently collected by adding protein A or G beads (1/10 volume, Sigma), fractionated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The blots were incubated with anti-phosphotyrosine (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-α-actin, anti-PLB antibodies, anti-phospho-Ser,^{16 (link)} or anti-phospho-Thr^{17 (link)} PLB antibodies (Badrilla, Leeds, UK) and subsequently incubated with goat anti-mouse or anti-rabbit alkaline phosphatase-conjugated secondary antibodies (Santa Cruz Biotechnology). The proteins were visualized using an enhanced chemiluminescence system (Intron, Seongnam, Republic of Korea) and an LAS 3000 imaging system (Fuji, Tokyo, Japan). To detect the total or tyrosine-phosphorylated α-actin in SR vesicles, we removed the IgG heavy chain band using ImmunoCruzTM IP/WB Optima E (Santa Cruz Biotechnology, Inc.) according to the manufacturer’s instructions.

Fresh LV tissues were embedded in the optimal cutting temperature compound (OCT) on dry-ice and then stored at -80°C. Before staining, LV tissues were cut into 10 um thick slices on a microtome-cryostat and mounted on super-frost plus slides. The tissue slides were then warmed at room temperature for 30 minutes and fixed in ice cold acetone for 5 minutes and then dried in the air. The following primary antibodies were employed: mouse anti-α smooth muscle actin (Sigma-Aldrich), mouse anti-α-actin (sarcomeric, Sigma-Aldrich) and anti-rabbit BKca β1 (Santa Cruz). After blocking with 5% bovine serum albumin(BSA) for 1 hour at room temperature and incubation with the primary antibodies at 4°C overnight, tissue slides were then incubated with the secondary antibodies raised against mouse and rabbit IgG conjugated with FITC and Texas Red (Santa Cruz) respectively for 1 hour at room temperature. After rinsing in PBS-Tween20 for several times, the slides were counterstained with DAPI for 1 minute and rinsed in PBS-Tween20 several times. The slides were then covered with an anti-fade mounting medium and visualized using EVOS FLc (Fisher).

The following antibodies were used:

mouse anti-Fmi (1:10; Developmental Studies Hybridoma Bank),

mouse anti-Fascin (1:10; Developmental Studies Hybridoma Bank),

mouse anti-Scar (1:50; Developmental Studies Hybridoma Bank),

rabbit anti-Dia (1:1,000, donated by P Adler),

mouse anti-Chic (1:10; Developmental Studies Hybridoma Bank),

rabbit anti-PatJ (1:500), mouse anti-HA (1:50; BioLegend),

mouse anti-α-actin (1:1,000; Sigma-Aldrich).

All fluorophore-conjugated secondary antibodies were used at 1:200 and obtained from Jackson ImmunoResearch Laboratories, Inc. Rhodamine-phalloidin was from Invitrogen and used at 1:400.