Mouse monoclonal anti myc 9e10

Adult ventricular cardiomyocytes were infected at an MOI of 5 as for live imaging and kept in storage solution in a humidified incubator at 37°C, 5% CO2. At 48 hours an aliquot was taken and paraformaldehyde to a final concentration of 4% was added, at 10 minutes, cells were pelleted (200rpm, 2min) in a benchtop centrifuge, washed with 1xPBS and resuspended in PBS. Cells were spun (Cytospin, Thermo Scientific) onto glass slides, and ringed with a PAP pen (Sigma). Permeabilisation with 0.1% Triton-X-100 in Tris Buffered saline for 10 minutes at room temperature was followed by blocking (0.2% albumin in permeabilisation buffer) for 20 minutes. Primary antibodies (mouse monoclonal 9E10 anti-myc (Santa-Cruz), and rabbit polyclonal anti-DsRed (Clontech) were diluted 1:200 in blocking buffer. Three hours after primary incubation cells were washed in permeabilisation buffer, and counter stained with Alexa-488 anti-mouse, and Alexa-568 anti-rabbit fab fragment secondaries (Invitrogen), nuclear counterstaining was with Topro3 (Invitrogen), for an hour before washing and mounting (Vectashield, Vector labs). Images were acquired on a Leica SP5 confocal microscope with a 63x oil immersion lens. hSC-CM’s were plated onto 0 thickness coverglass, infected at an MOI of 5. Cells were fixed and stained 48 hours after infection as above.

Cells were washed once with PBS and resuspended in NP-40 lysis buffer (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonylfluoride, 10 mM NaF, 0.5 mM sodium orthovanadate, leupeptin (10 µg/ml), aprotinin (10 µg/ml), 1% (v/v) NP-40 and 10% (v/v) glycerol). After incubation for 20 min on ice, cells were centrifuged for 10 min at 13000 rpm to remove the insoluble fraction and the supernatant was transferred to a new tube. SDS sample buffer was added and the extracts were heated for 5 min at 95 °C, followed by separation of proteins using SDS-PAGE and Western blotting as previously described^{73 (link)}. The following antibodies were used for immunoblotting: rabbit polyclonal anti-CNOT1 (kind gift of Dr. Elisa Izaurralde), mouse monoclonal (2191C2a) anti-CNOT2 (Santa Cruz), rabbit polyclonal anti-CNOT3 (self-made), mouse monoclonal (9E10) anti-Myc (Santa Cruz) and mouse monoclonal (Tub2.1) anti-Tubulin (Sigma Aldrich).