Immobilon western chemiluminescence hrp substrate

At the appropriate time point for each experiment, whole-cell lysates were prepared using a radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technologies, Beverly, MA, USA) with a cocktail of phosphatase inhibitors (Thermo Fisher Scientific, San Jose, CA, USA). The protein concentrations were determined using a Bradford assay (Sigma-Aldrich, St. Louis, MO, USA). For the Western blot analysis (reducing system), the samples were diluted in 5× Laemmli buffer (300 mM Tris-HCl pH 6.8, 10% SDS (w/v), 5%, 2-mercaptoethanol, 25% glycerol (v/v), and 0.1% bromophenol blue (w/v)) and boiled for 5 min. For the non-reducing PAGE conditions, the samples were diluted in 5X Laemmli buffer without 2-mercaptoethanol and boiled for 5 min. Proteins (20 μg) were separated using 8–15% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare Life Sciences, Madison, WI, USA). The nonspecific binding sites on the PVDF membranes were blocked with 5% non-fat milk in TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 1% Tween-20). The membranes were then hybridized with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 24 °C. The membranes were then developed using Immobilon Western Chemiluminescence HRP substrates (Merck Millipore, Billerica, MA, USA).

Proteins (20 μg), from cell lysates, were separated by 8–12% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Biotrace^TM, PALL Life sciences, Ann Arbor, MI, USA). The sample membrane was immunoblotted with primary and secondary antibodies for at least 16 h. Signals from the antibody–protein conjugates were detected by the chemiluminescence kit (Immobilon Western Chemiluminescence HRP Substrates, Merck-Millipore, Billerica, MA, USA). The images of the relative bands were analyzed by the luminescence/fluorescence imaging system (GE healthcare) and MultiGauge image analysis software version 3.0 (Fujifilm, Stockholm, Sweden). The α-tubulin antibody was purchased from Invitrogen^TM, Thermo Fisher Scientific (Carlsbad, CA, USA). Antibodies against xCT, SIRT3, AMPK, p-AMPK, acetyl-CoA carboxylase (ACC), p-ACC, and acetylated-lysine Ac-K-103 were purchased from Cell Signaling Technology (Beverly, MA, USA).

Cells were lysed in radioimmunoprecipitation assay lysis (RIPA) buffer. The cell lysate was collected after centrifugation at 13,000× g for 15 min. Proteins (20 μg) from cell lysates were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (BiotraceTM, PALL Life sciences, Ann Arbor, MI, USA (BSP0161)). Signals after immunoblotting with primary and secondary antibodies overnight on the sample membrane were detected by a chemiluminescence kit (Immobilon Western Chemiluminescence HRP Substrates, Merck-Millipore, Billerica, MA, USA (WP20005)). A luminescence/fluorescence imaging system (GE Healthcare) and multi-gauge image analysis software version 3.0 (Fujifilm, Stockholm, Sweden) were used to analyze the image. The preparation of the RIPA buffer and detailed procedure has been described in a previous study [27 (link)]. The antibodies against α-tubulin (62204), p-eIF2α (44728G), eIF2α (AHO0802) (InvitrogenTM, Thermo Fisher Scientific, Carlsbad, CA, USA), ATF4 (Proteintech, Rosemont, IL, USA (10835-1-AP)), and xCT (Cell Signaling Technology, Beverly, MA, USA (#12691)) were used in this study.

Cell lysate was extracted using radioimmunoprecipitation assay buffer (RIPA buffer: 50 mM Tris-HCl buffer, pH 7.5, containing 0.15 M NaCl, 0.5 % sodium deoxycholate, 0.5 % SDS, 0.1% Triton X-100, 10 μg/ml aprotinin, 2 mM ethylenediaminetetraacetic acid [EDTA], 2 mM Na₃VO₄, and 1 mM PMSF). The concentration of protein was determined using Bradford reagent with BSA as the standard. Twenty micrograms of lysate protein were resolved by 8-12 % SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (PVDF membrane, Biotrace™, PALL Life sciences, Ann Arbor, MI, USA), and immunoblotted with antibodies. The protein contents were visualized using a chemiluminescence kit (Immobilon Western Chemiluminescence HRP Substrates, Merck-Millipore, Billerica, MA, USA). The images of Western blot were observed using a Luminescence/Fluorescence Imaging System (GE Healthcare), and the signal intensities were quantified using Multi Gauge image analysis software (Fujifilm).

Neutrophils (5 × 10⁶/ml) were left unstimulated or were stimulated with iIC/PMA for 15 min at 37°C in medium with different pH values. In some experiments, neutrophils were preincubated with various inhibitors. Whole cell lysates were prepared using TCA as described (60 (link)). Western blot analysis was carried out by using antibodies against human phospho Akt (Thr308), phospho-p44/42 MAPK (ERK1/2, thr202/Tyr204), phospho p38 MAPK (Thr180/Tyr182), or beta-actin (all from Cell Signaling Technology) and probed with HRP-conjugated anti-rabbit or anti-mouse IgG (New England Biolabs, USA). The signals were detected by using Immobilon Western Chemiluminescence HRP substrate (Millipore, USA) by using the Fusion Fxt Chemiluminescence reader (Vilber Loumat, Germany). Signals of pAkt, pERK1/2, or pp38 were normalized to beta-actin signals on the same blots or of the same sample by using ImageJ software (NIH, USA) (61 ).

Tissues and cells were washed with precooled PBS and treated with RIPA buffer (Beyotime, Shanghai) on ice for 30 min. Total protein was collected from the cell lysates and centrifuged at 4 °C and 12000 rpm for 20 min, and the supernatants of all lysates were reserved. Then, we determined the protein concentration of all samples by BCA methods (Beyotime). Loading buffer was added to the protein samples, and they were heated at 4 °C for 10 min. Then, all samples were ready for use in further studies. After a SDS–PAGE gel (Beyotime) was prepared, 25 μg of protein sample was added per well for each sample. By electrophoresis, the proteins with different molecular weights were separated in the gel, and then they were transferred to a PVDF membrane (Millipore, USA). The membrane was incubated with a primary antibody at 4 °C overnight. On the following day, after being washed with TBST (Beyotime) 3 times, the PVDF membrane was incubated with an HRP-conjugated secondary antibody for 1 h at room temperature on an orbital shaker. Finally, the PVDF membrane was exposed to light for imaging with immobilon western chemiluminescence HRP substrate (Millipore), and the relative intensity of the protein band was determined with Image-Pro Plus software version 6.0 (Media Cybernetics, Rockville, MD, USA).