Pierce 660 nm reagent

Cells were firstly lysed using lysis buffer (Nanjing Jiancheng, Beijing, China). The protein concentrations were measured using Pierce™ 660 nm reagent (#22660, Thermo Fisher Scientific, USA). Same amount of protein (20 μg) was loaded for 10% SDS-PAGE, and transferred to a PVDF membrane. TBST containing 5% non-fat milk was used to block membrane for 2 h. Then, the proteins were incubated with related primary antibodies (Rabbit polyclonal to CCN1, ab230947; Rabbit monoclonal to GAPDH, ab9485) at 4°C overnight. After washing with PBS, the membrane was incubated with secondary antibodies for 2 h. Then, membrane was measured using an enhanced chemiluminescence detection kit (#34580, Thermo Fisher Scientific, USA), and ImageJ software was used to analyze protein band.

After ROR1 inhibitor treatment (10 µM) and vehicle (DMSO) control treatment, the cells were collected by scraping. The proteins were lysed using RIPA buffer (150 mM NaCl, 1.0% Triton-X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with 1 mM PMSF (Protoc, 2017 ). Bradford Assay measured cell lysates’ protein concentration using Pierce 660 nm reagent (Thermofisher, #22660). The Protein lysates were processed by mixing with laemmli buffer at a 1:1 ratio, followed by boiling. Equal amounts of proteins were loaded into the wells of an SDS-PAGE gel, followed by electrophoresis. The proteins were transferred to a nitrocellulose membrane, then blocked for 30 min in 5% bovine serum albumin. The membrane was incubated overnight at 4°C in anti-ROR1 antibody (Cell signaling, #D6T8C), then washed with TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween 20), then incubated in horseradish-peroxidase-linked anti-Rabbit IgG (Invitrogen, #65-6120) for 1 h at room temperature. Protein bands were imaged by chemiluminescence. GAPDH (Cell signaling, #D16H11) or β-actin (Santa Cruz Biotechnology, #sc47778) were loading controls.

After sacrifice, the brains of rats were taken under low temperature and the hippocampus was isolated on ice. The left hippocampus was put into the cryopreservation tube and stored at −80°C. The tissues were homogenated and centrifuged first. Lysis buffer (RIPA: PMSF = 100:1) was used to further lysed tissues. After centrifugation at 12000 r/min for 15 min, the protein supernatant was isolated, and protein content was measured using Pierce^™ 660 nm reagent (Thermo Fisher Scientific, USA). 10% SDS-PAGE was performed to isolate protein, and the target proteins were transferred to a PVDF membrane. TBST with 5% non-fat milk was applied to incubate membranes for blocking. Primary antibodies incubation was performed at 4°C overnight. Quantity One software was used to analyze the gray value of protein bands. The following antibodies were used in this study. Rabbit anti-Bcl-2 (1:1000, ab32124, Abcam, UK), Rabbit anti-Bax (1:1000, ab32503, Abcam) Rabbit anti-Cleaved caspase-3 (1:1000, ab32042, Abcam), Rabbit anti-LC3 (1:500, ab63817, Abcam), Rabbit anti-GAPDH (1:500, ab245355, Abcam), Rabbit anti-AMPK (1:500, ab32047, Abcam), Rabbit anti-p-AMPK (1:500, ab133448, Abcam), Rabbit anti-mTOR (1:1000, ab134903, Abcam), Rabbit anti-p-mTOR (1:1000, ab109268, Abcam), Rabbit anti-P62 (1:1000, ab109012, Abcam).

Particles (300 µg) with bound proteins were washed sequentially for 30 min with different buffers: (a) 50 mM HEPES 0.1% OGP pH 7, (b) 100 mM NaAc 0.1% OGP pH 5. Each sample was shaken in a laboratory tube rotator with 2 mL of the corresponding buffer solution for 30 min and was centrifuged 10 min at 4 °C in a microcentrifuge. The supernatant was collected and stored at −80 °C until analysis.
The eluted proteins in each step of the purification process were precipitated by a methanol/chloroform protocol, [45 (link)] quantified by Pierce 660 nm reagent (Thermo Scientific) in 8 M urea, 25 mM ammonium bicarbonate ((NH₄)HCO₃), reduced with 50 mM of Tris(2-carboxyethyl) phosphine (TCEP), alkylated with 20 mM of methyl methanethiosulfonate (MMTS) and finally digested with trypsin (1:20 enzyme:protein, weight ratio) according to Reference [46 (link)]. The digest was passed through a SEP-PAK C18 column prior to analysis by mass spectrometry (Waters, Milford, MA, USA).
The samples digested were subjected to electrospray ionization mass spectrometry, 1D-nano LC ESI-MSMS analysis using a nano-liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, AB SCIEX, Foster City, CA, USA) coupled to a high-speed Triple TOF 5600 mass spectrometer (AB SCIEX, Foster City, CA, USA) with a Nanospray III source. Full description of the methodology has been published elsewhere [47 (link)].

Monolayer of cells seeded in 6-well plates were treated with RvD2 (100 nM) and vehicle for 24 h, then lysed using lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM Na3VO4, 1 mM PMSF, 100 mM NaF, 1% Triton x-100) with the addition complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein concentration was then determined by Pierce 660 nm Reagent (Thermo Fisher Scientific, Waltham, MA, USA; Catalog # 22660) with BSA as standard. Placental cell lysates containing 30 ug of protein were resolved in SDS-PAGE and the proteins were transferred onto nitrocellulose membrane (BioRad Laboratories, Hercules, CA, USA) for immunodetection using anti-GPR 18 antibody as described [44 (link)].