Supersignal west dura extended duration substrate chemiluminescent substrate

MP2 and scorpine protein synthesis in midgut and salivary glands of the transgenic lines was evaluated by Western blot. Also, 5 midguts and 10 salivary glands were dissected in PBS and placed in microtubes containing RIPA Buffer (Thermo Fisher Scientific), 1% Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), and 0.1 mM PMSF (Sigma-Aldrich). Samples were homogenized and stored at –70°C. An equivalent of 0.25 midgut and 5 salivary glands were resolved in a NuPAGE 10% Bis-Tris Protein Gel (Invitrogen) under reducing conditions and transferred to a PVDF membrane Invitrogen Power Blotter Select Transfer Stacks. After the transfer, the membrane was washed with TBST 1% (Sigma-Aldrich), incubated with blocking buffer (5% milk powder in TBST 1%) overnight at 4°C, and probed with mouse anti-MP2 or anti-scorpine at a 1:1000 dilution in TBST 1% overnight at 4°C. The membrane was washed and incubated with an anti-mouse HRP-linked antibody (Cell Signaling) at a 1:10,000 dilution in TBST 1% for 2 hr at room temperature. Detection was done with the SuperSignal West Dura Extended Duration Substrate Chemiluminescent Substrate (Thermo Fisher Scientific) and imaged using an Azure Imager c600 (Azure Biosystems).

huPAI-1 protein synthesis in midgut and salivary glands of the transgenic lines was evaluated by Western blotting. A pool of five midguts and ten salivary glands were dissected in PBS (0.1 M, pH 7.8), collected in microtubes containing RIPA Buffer® (Thermo Fisher Scientific), 1% Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific), and 0.1 mM PMSF (Sigma-Aldrich). Samples were homogenized and stored at −70 °C. An equivalent of five midguts (~40–50 µg of protein) and five salivary glands (~10 µg of protein) were resolved in a NuPAGE™ 10% Bis-Tris Protein Gel (Invitrogen) under reducing conditions and transferred to a PVDF membrane Invitrogen™ Power Blotter Select Transfer Stacks (Invitrogen). After the transfer, the membrane was washed with TBST 1% (Sigma-Aldrich), incubated with blocking buffer (5% milk powder in TBST 1%) overnight at 4 °C, and probed with a mouse anti-human PAI-1 antibody (BD Biosciences, clone 41/PAI-1, #612024) at a 1:1000 dilution in TBST 1% overnight at 4 °C. The membrane was washed and incubated with a rabbit anti-mouse HRP-linked antibody (Cell Signaling, # 7074) at a 1:10,000 dilution in TBST 1% for 2 h at room temperature. Detection was done with the SuperSignal™ West Dura Extended Duration Substrate Chemiluminescent Substrate (Thermo Fisher Scientific), and imaged using an Azure Imager c600^® (Azure Biosystems).