Alexa 647

Cardiomyocytes were plated on glass-bottom dishes (MatTek Corp., Ashland, MA, USA) for immunofluorescence and confocal imaging. For immunofluorescence analysis, isolated cardiomyocytes were fixed with 4% paraformaldehyde (PFA) for 30 minutes at 4 degrees Celsius. Next, cardiomyocytes were incubated with permeabilization buffer (0.5% Triton X-100, 0.2% Tween-20 in PBS) for 30 minutes at 4 degrees Celsius. Blocking buffer (5% FBS in permeabilization buffer) was then added and incubated for 30 minutes at room temperature. Primary antibodies (listed above) were then added (SERCA2A – 1:500, PLN – 1:1000, RyR2 – 1:1000, DHPR – 1:700). Cardiomyocytes were then incubated with primary antibody overnight at 4°C and fluorophore-conjugated secondary antibody staining (Alexa 647, Molecular Probes) was performed at room temperature for 1 hour in the dark. Nuclear counterstaining was performed using 1 μg/ml Hoechst 33342 (Cell Signaling, #4082) at room temperature for 15 minutes in the dark.

Staged embryos and third instar larval samples were prepared and stained using standard protocols [35] (link). The following primary antibodies were used: guinea pig anti-Slik[43] (link), mouse anti-SRF (1∶200, DSHB), rat anti-E-Cadherin (1∶200, DSHB), rabbit anti-Dof [71] (link), antibody against phosphorylated moesin is rabbit anti P-ERM (1∶200, S3149 Cell Signaling), rabbit anti-Moesin (1: 1000, François Payre), rabbit anti-pMoesin (1: 1000, François Payre). Secondary antibodies: Alexa 468 and Alexa 568 and Alexa 647 (Molecular Probes) were used at a dilution of 1∶2000.

The following primary antibodies were used: guinea-pig anti-Tj (G5 or GP6, 1:5000) [57 (link)], rat anti-Bab2 (R10, 1:3000; or R7, 1:2000) [20 (link)], rabbit anti-Vasa (1:2000) [80 (link)], rabbit anti-Vasa (d-260, 1:500; Santa Cruz Biotechnology), chicken anti-Vasa (1:5000; gift from K. Howard and M. Van Doren), rabbit anti-α-spectrin (#254, 1:1000; gift from D. Branton), mouse anti-LamC (LC28.26, 1:50), mouse anti-Hts (1B1, 1:5), mouse anti-N (C17.9C6, 1:5; C458.2H, 1:5), mouse anti-Dl (C594.9B, 1:5), mouse anti-Engrailed (4D9, 1:5), and mouse anti-ßPS integrin (CF.6G11, 1:10) (Developmental Studies Hybridoma Bank), rabbit anti-pMad (PS1, 1:250; gift from T. Tabata) [81 (link)], rabbit anti-pMad (pSmad1/5, 41D10, 1:100; Cell Signalling), rabbit anti-ß-galactosidase (1:1500; MP Biomedicals), and rabbit anti-GFP (1:100; BD Biosciences). Secondary antibodies (1:400) were conjugated either to Cy3, Cy5 (Jackson Immuno Research Laboratories), Alexa-405, Alexa-555, Alexa-488, or Alexa-647 (Molecular Probes, Life Technologies). Ovaries were mounted in Vectashield (Vector Laboratories).

HEK293T cells were plated on 6 well plates for 24 h prior to use. Human E-cadherin-GFP and inducible zebrafish WT or mutant march8 plasmids were transfected into the cells (see above). Cells were incubated for 12 h and then transferred to fresh media containing 50 µM tebufenozide for 8 h. After incubation, the cells were harvested and non-permeablized cells were stained at 4°C with extracellular domain-specific E-cadherin antibody (67A4, Santa Cruz, 1∶100) for 30 min in FACS buffer (0.5% BSA, 1% Sodium Azide in PBS). Cells were washed 3 times with FACS buffer and were stained with Alexa-647 (1∶1000, Molecular Probe) secondary antibody at 4°C for 30 min. Data acquisition was performed on a FACScan flow cytometer (BD Bioscience), and data were analyzed using FloJo software.

293T cells (5 × 10⁵) were cotransfected with 0.8 µg of either the control vector and 200 ng of pCa-EGFP, or the combination of 120 ng of either the MARCH expression plasmid (WT or a tyrosine mutant) or the control vector, 20 ng of either pC-VSVg or pC-NLenv, 500 ng of pNL-Luc2-E(-), and the control vector up to 1 μg. To analyze cell-surface expression, the transfected cells were incubated with either anti-HIV-1 gp120 goat polyclonal antibody (Abcam, ab21179) or anti-VSV-G mouse monoclonal antibody (Sigma-Aldrich, V5507), followed by staining for 30 min on ice with either a rabbit anti-goat IgG conjugated with Alexa 647 (Molecular Probes, A21446) or a goat anti-mouse IgG conjugated with R-phycoerythrin (Molecular Probes P-852), respectively. Cells were washed extensively with PBS with 4% FBS and fixed with 4% formaldehyde in PBS. To analyze intracellular expression, the transfected cells were fixed with 0.01% formaldehyde in PBS, permeabilized with 0.05% saponin for 10 min, and immunostained with the antibody against either gp120 or VSV-G, followed by incubation with secondary antibodies, as described above. GFP-positive cells were sorted and analyzed for the expression of HIV-1 Env or VSV-G by flow cytometry using a BD FACS Canto II, and the data were collected and analyzed with BD FACS Diva Software.