Azure c600 imaging system

The nitrocellulose membranes with transferred proteins were blocked for one hour with 5% non-fat milk (Blotting Grade Blocker Non-Fat Dry Milk, Bio-Rad) in PBS-Tween (0.5% Tween 20; Sigma-Aldrich) and incubated in parallel with primary antibodies anti-MSMB (1:500 dilution, polyclonal rabbit antibody) and anti-ubiquitin antibody (FK2, 1:250 dilution, monoclonal mouse antibody recognizing mono- and polyubiquitinated conjugates; ENZO Life Sciences), both in 1% non-fat milk in PBS-Tween, overnight. For protein normalization purposes, the membranes were stripped and incubated with monoclonal antibody anti-alpha-tubulin DM1A (1:5000 dilution; Sigma-Aldrich). The following day, membranes were washed in PBS-Tween and incubated with HRP-conjugated species-specific secondary antibodies such as goat anti-rabbit IgG and goat anti-mouse IgG (1:3000 dilution; Bio-Rad) in 1% non-fat milk in PBS-Tween for 60 min at laboratory temperature. The membranes were washed four times in PBS-Tween and two times in PBS, reacted with a chemiluminescent substrate (Super Signal West Pico Chemiluminescent Substrate; ThermoFisher Scientific), and reactive bands were screened with Azure c600 imaging system (Azure Biosystems).

Embryos were deyolked in batches as outlined in Link et al. [20 (link)]. Briefly, 15–20 larvae were placed in a 1.5ml Eppendorf tube. Egg water was removed and 1 ml deyolk buffer (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO₃) was added. Larvae were pipetted through a p200 pipette tip to disrupt the yolk, and then agitated at 1100 RPM for 5 minutes. They were then centrifuged at 300g for 30 seconds. The larvae were then washed twice by removing the deyolk buffer, adding 1mL wash buffer (110 mM NaCl, 3.5 mM KCl, 2.7 mM CaCl2, 10 mM Tris-Cl pH8.5), agitating at 1100 RPM for for 2 minutes, and centrifuging at 300g for 1 minute. After washes, all liquid was removed, and 4 ul 1x SDS buffer (5% 2-Mercapto Ethanol, 2% SDS, 5% glycerol, 0.05 mM Tris pH 6.8, 0.017% Bromophenol Blue in water) per larva was added. The samples were homogenized and 6 larval equivalents of protein isolate was assayed. The blots were labeled with a rabbit-anti-pde6c polyclonal antibody (Abcam ab198744) and a mouse-anti-alpha tubulin monoclonal antibody (Sigma T6199). A HRP-conjugated goat-anti-rabbit polyclonal antibody (Abcam ab6721) and a HRP-conjugated goat-anti-mouse polyclonal antibody (Abcam ab97265) were used as secondary antibodies. The HRP label was imaged using a SuperSignal (TM) West Pico PLUS Chemiluminsecent Substrate (Thermo Scientific 34579) and an Azure c600 imaging system (Azure Biosystems).

The protein samples were mixed with 4× lithium dodecyl sulfate sample buffer (Invitrogen, Carlsbad, CA, USA) and heated at 90 °C for 10 min. Samples were run on 4–12% gradient bis–tris gels (Invitrogen) and then transferred to a nitrocellulose membrane using the iBlot2 gel-transfer device (Invitrogen). The membranes were blocked in buffer [PBST; 10 mM sodium phosphate, 132 mM NaCl, 2.7 mM KCl, and 0.05% Tween-20, pH 7.4] for 1 h at 25 °C with shaking, washed three times with PBS-T for 10 min, and then incubated with a home-made primary antibody against FMDV VP1 (76.5E) and 3B (4G24) diluted 1/2000 in PBST at 4 °C overnight. The membranes were washed three times with PBS-T and incubated with HRP-conjugated goat anti-mouse secondary antibody (Invitrogen) diluted 1/4000 in PBST for 1 h at RT. Proteins were detected with Pierce ECL Substrate (Invitrogen) using an Azure C600 imaging system and cSeries Capture Software (Azure Biosystems, Dublin, CA, USA).