Bca protein assay kit

Frozen tissue samples and Siha and Hela cells were processed for protein extraction and western blotting using standard procedures. In brief, 100 mg of tissue (homogenized first using a homogenizer) and cells were washed twice in phosphate-buffered saline (PBS) and then lysed in radioimmunoprecipitation assay (RIPA) buffer containing 100 mM phenylmethanesulfonyl fluoride (PMSF, Solarbio) and protease inhibitor cocktail. Total protein in the supernatant was quantitated by the bicinchoninic acid (BCA) protein assay kit (BCA protein assay kit, Thermo Scientific). The proteins were resolved by using 8% SDS-PAGE and transferred onto PVDF membranes, which were incubated overnight with primary antibodies (anti-PAF1, 15,441–1-AP, Proteintech; anti-IER5, ab59133, Abcam) at 4 °C. The membranes were then probed with secondary antibodies. Finally, the western blotting bands were exposed using an Amersham Imager 600 (General Electric Company, USA) with an ECL chemiluminescence kit (Millipore Corporation, Billerica, MA, USA). The Western blotting bands were quantified using ImageJ software and normalized to the level of GAPDH.

Cells were harvested and washed twice with cold PBS, and lysed using the radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) containing 10 nM phenylmethanesulfonyl fluoride (PMSF). Protein concentrations were determined by a BCA protein assay kit (ThemoFisher Scientific). Samples were separated with sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto a polyvinylidene fluoride (PVDF) membrane (Millature Antiipore, Billerica, MA). Membranes were blocked with 5% skim milk for 1 h, and incubated with primary antibody at 4 °C overnight followed by secondary antibody for 1 h at room temperature. Antibodies included β-actin (1:2000, Beyotime), Rictor (1:1000, Abcam), Hif1α (1:1000, Abcam), Glut1 (1:1000, Abcam), p-AKT (1:1000, Abcam), AKT (1:1000, Abcam), p-FoxO1 (1:1000, CST), FoxO1 (1:1000, CST), HRP-conjugated goat anti-rabbit IgG (Genshare, Xian, China), and HRP-conjugated goat anti-mouse IgG (Genshare, Xian, China).

Rats were anesthetized by inhalation of isoflurane followed by cardiac perfusion with ice-cold PBS. The hippocampus was collected quickly, and the dorsal portion of the hippocampus was homogenized in 300 µl ice-cold lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 12 mM beta-glycerophosphate, 1% Triton) with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The lysate was then sonicated for 30 s and centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was used for ATP detection (Firefly Luciferase Bioluminescence Assay, Invitrogen). Protein concentration was measured by a BCA protein assay kit (Themo Fisher Scientific, Waltham, MA), and 5 µg of total protein was assayed for measuring ATP. ATP concentration was quantified according to the standard curve.

The total protein of hypothalamus from each mouse was extracted using RIPA Lysis buffer (Biomiga, Santiago, CA, USA) and the BCA protein assay kit (Termo Fisher Scientific) was used to determine the protein concentration. The protein expressions of apelin and APJ were measured by western blot and the procedure was performed as previously described [56 (link)]. The 12% SDS-PAGE was selected to conduct transmembrane according to relative molecular weight of apelin and APJ, and then proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. PBST with 5% nonfat milk was used to block the membranes, and the primary antibodies were apelin (M-77) (SC-33805, 1:100 dilution, Santa Cruz, Dallas, TX, USA), Apelin receptor Antibody (APJ) (PA5-21306, 1:500 dilution, Thermo Scientific) and β-Tubulin Monoclonal Antibody (A01030, 1:5000 dilution, Abbkine, Inc., Redlands, CA, USA). The SuperSignal^® West Pico Chemiluminescent Substrate (Thermo Scientific) was used to develop the membranes and the Tanon-5200 analysis system (Tanon, Shanghai, China) was used for exposure, then the optical density of protein bands were read by Tanon Gis software.

Protein samples were prepared using the BCA Protein Assay Kit (Thermal Fisher). A total of 50 μg of protein was separated on 10% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h before being incubated overnight at 4° C with primary antibodies against PFN2 (Abcam), CD31 (Abcam), TSG101 (Abcam), Alix (Proteintech), Smad2 (Huabio), Smad3 (Immunoway), AKT (CST), p-AKT (CST), ERK (CST), p-ERK (CST), PI3K (CST), or GAPDH (CST). The membranes were washed three times with PBST and then incubated with HRP-conjugated secondary antibodies (Yeasen) for 1 h. After the final wash with TBST, the proteins were detected using enhanced chemiluminescence reagents.

NAGLU activity levels in cells plated in 96-well plates, as described above, was determined using the 4MU substrate, 4-Methylumbelliferyl-N-acetyl-α-D-glucosaminide. Briefly, cells in each well were washed extensively then lysed at room temperature for 15 minutes with 57.5 uL of mammalian protein extraction reagent per well (M-PER; ThermoFisher Scientific, Waltham, MA). Of this lysate, 10 uL was used to determine total protein levels using a BCA protein assay kit (ThermoFisher Scientific, Waltham, MA). The remaining lysate in each well was then incubated in the presence of 2 mM 4-Methylumbelliferyl-N-acetyl-α-D-glucosaminide in 0.2 M Acetate buffer pH 4.8 for 30 minutes at 37°C in a total reaction volume of 85 uL. The reactions were terminated by addition of 200 uL Glycine/NaOH buffer pH 10.7 to each well and plates were read at Ex360 Em460 with 455 cut off using a Spectramax i3 plate reader (Molecular Devices). A standard curve containing known amounts of 4-Methylumbelliferone (4-MU Standard; Sigma-Aldrich, St Louis, MO) was included in each assay to calculate NAGLU activity levels. In some instances where indicated, 4 uM IGF2 (Cell Sciences, Canton, MA) or 5 mM mannose-6-phophate (Man6P; Sigma-Aldrich, St Louis, MO) was added to uptake medium.