Ab97265

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).

Cells were first trypsinized and resuspended in HLB buffer with protease inhibitors. After homogenization, cells were mixed with 1× SDS lysis buffer and boiled. Equal amount of protein from each sample was then separated using electrophoresis and transferred into nitrocellulose membranes. Membranes were then incubated with 5% skimmed milk, followed by overnight incubation with β‐catenin primary antibody (1:500, ab32572, Abcam), STAT3 primary antibody (1:1000, ab119352, Abcam), WIF‐1 primary antibody (1:1000, ab186845, Abcam), E‐cadherin primary antibody (1:1000, ab1416, Abcam), N‐cadherin primary antibody (1:1000, ab18203, Abcam), or GAPDH primary antibody (1:2000, ab9485, Abcam). This was followed by incubation with secondary antibody (1:2000, ab6721 or ab97265, Abcam, USA) for 2 hours. The protein bands of interest were visualized by an ECL Advanced Western Blot Detection Kit.

Serial 6.25 fold dilutions of each sE2 supernatant beginning with a 1 to 40 dilution were immobilized onto ELISA wells pre-coated with 500 ng Galanthus nivalis lectin (Sigma-Aldrich) and blocked with PBS containing 0.5% Tween 20, 1% nonfat dry milk, and 1% goat serum. Wells were probed with 0.5 μg of a mouse monoclonal anti-6x His-tag antibody (ab18184, Abcam) and quantified using a HRP-conjugated goat anti-mouse IgG secondary antibody (ab97265, Abcam). The EC50 for each sE2 construct was calculated by nonlinear regression analysis and fold differences in EC50 used to normalize sE2 concentration in subsequent experiments.