For experiments in which we froze actin assembly and disassembly with the JLY cocktail, cells were allowed to spread on PLL coated glass for 30 min in the presence of 10 µM Rock kinase inhibitor (Y27632).
After washing twice with ice-cold PBS containing protease inhibitor cocktail, cells were collected in lysis buffer (1% SDS, 10 m M EDTA, and 50 m M Tris, pH 8.1), incubated for 30 min on ice, and then sonicated to shear DNA.
Cells and total proteins from the cells were collected using cell lysis buffer supplemented with protease inhibitor cocktail (Cell Signaling #9803, Roche complete mini #1836153) after 30 minutes, 1 hour, and 2 hours of insulin stimulation for the low-dose T pre-exposed cells, and after 10, 20 and 30 min of insulin stimulation for the high-dose T treated cells.
After in vitro transcription from the recombinant plasmid, HCT-fused to a C-terminal FLAG peptide (HCT-FLAG) was in vitro synthesized in the wheat germ cocktail (CellFree Sciences, Japan) according to the manufacturer's instructions.
Briefly, EE2 ECs or excised cremaster muscles were harvested using ice-cold IP lysis buffer containing complete protease and phosphatase inhibitor cocktail (Cell Signaling) and the samples were sonicated on ice three times for 5 seconds each.
For Western Blot of P1-tdTomato from erythrocytes, 20 μl of packed erythrocytes was lysed in ice-cold RIPA buffer (G-Biosciences, St . Louis MO) containing 1 100 Protease Inhibitor cocktail (Cell Signaling Technologies, Danvers, MA).
After centrifugation, cells were washed in 10 mL ice-cold PBS, and re-suspended in 100 μL of PBS supplemented with 1% protease inhibitor cocktail (Cell Signaling, #5871S), 1% phosphatase inhibitor 2 (Sigma Aldrich, #P5726), 1% phosphatase inhibitor 3 (Sigma Aldrich, #P0044), and 10% Cell Lysis Buffer (Cell Signaling, #9803).
However, following the addition of a hormonal cocktail, the cells undergo further cell divisions.
After the co-culture of uninfected MDDC with CFSE-labelled MVA-B-infected MDDC (see below) during 48 h with maturation cocktail, the cells were washed with PBS and stained with PE-anti-CD80 antibody (BD Biosciences) at 4°C.
After a two-week treatment with the CRFVPT cocktail, beating cells were also found.
In the absence of this chemical cocktail the cells retain a more primitive fibroblastic phenotype.
