Membrane protein extraction kit

Total cellular proteins were extracted using 1× cell lysis buffer (Cell Signaling Technology). Membrane proteins were isolated using a membrane protein extraction kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations were determined using the Bradford protein assay according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Proteins were separated by 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk blocking buffer for 1 h, followed by overnight incubation at 4 °C with primary antibodies in blocking buffer. The membranes were washed in washing buffer three times, followed by incubation with secondary antibodies for 1 h. After successive washes, the membranes were developed using a SuperSignal West Pico Chemiluminescent kit (Thermo Fisher Scientific).

For total protein collection, cells were harvested and lysed using RIPA buffer (50 mM Tris–HCl, pH 8.0; 150 mM NaCl; 5 mM EDTA; 0.1% SDS; and 1% NP-40) supplemented with protease inhibitor cocktail^{26 (link)}. For secreted Samd1 collection, the culture medium containing the Samd1 was filtered through a 0.2 μm membrane and then concentrated by protein concentrator (Thermo, 10 K, 88527). Membrane protein extraction was performed with Membrane Protein Extraction Kit (Thermo, 89842) according to manufacturer’s instructions.
The protein concentration was determined by the Bradford assay, and equal amounts of protein in the lysates were boiled and fractionated by 7–12% SDS-PAGE. Primary antibodies against the following proteins were used: ApoB, β-actin, LaminA, Cadherin (Proteintech), Samd1 (Abcam). Signal was detected using Western ECL Substrate (Bio-Rad). To measure signals from the same gel, some full-length original blots might not be able to obtained.

The transfected cells were grown to 80% confluence in 12-well plates. The experiment was divided into three parts: (1) Cells were incubated with CAP (25 μM) alone, or in combination with SB-705498 (0.1 μM), or in combination with RR (10 μM), or in combination with NOVO (1, 5, and 10 μM), for 5, 15, 30, and 60 min; (2) Cells were incubated with CAP (25 μM) for 5, 15, 30, and 60 min at the beginning. SB-705498, RR or NOVO was then added to the medium for another 15, 30, and 60 min. (3) After pretreatment with SB-705498, RR or NOVO for 24 h, cells were incubated with CAP (25 μM) in culture medium at 37°C in 5% CO₂ for 5, 15, 30, and 60 min, respectively. After incubation, the medium was removed, and cell layers were washed three times with ice-cold PBS and then extracted with Membrane Protein Extraction Kit (Thermo Fisher Scientific Inc, Rockford, IL, United States), according to the manufacturer’s protocol. The cytosolic protein extracts were stored at -80°C until LC-MS/MS analysis.

Female Ae. aegypti were fed a blood meal and 72 h later, after the blood was digested, guts were dissected out in a drop of saline and transferred into an Eppendorf tube containing 100 mM PBS pH 7.2 (1.0 mL) and 100 μL of Protease inhibitor cocktail (Thermo Fisher Corporation, Waltham, MA USA). The tube was centrifuged for 5 min at 5000 rpm using an Eppendorf desk centrifuge, the supernatant removed, and the pellet was thoroughly homogenized with a Teflon tip. The homogenate was recentrifuged at 5000 rpm, the supernatant removed, and the pellet extracted using a Membrane Protein Extraction Kit (Mem-PER Thermo Fisher, USA) following manufacturer instructions. After extraction, the solubilized membrane proteins were centrifuged at 15,000 rpm for 3 min and the supernatant dialyzed against 0.1 M PBS pH 7.2 (100 mL) containing 0.5% CHAPS for 3 h at 4 °C in a dialysis bag with M_r cutoff of 3.5 kDa. The dialysis buffer was replaced, and the dialysis was repeated one more time. After dialysis, 50 μL of protease inhibitor cocktail was added to the extracted membrane protein (0.75 mL, 3.5 mg) and the extract stored at −80 °C.