Cellytic m lysis buffer

Western blotting was performed as previously described [27 (link)]. Ca9-22 cells were lysed in CelLytic M lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitor cocktail (Nacalai Tesque). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories), and probed with rabbit anti-human ANGPTL2 polyclonal antibody (Proteintech Group, Chicago, IL, USA, Cat:12316-1-AP) or rabbit anti-human β-actin monoclonal antibody (Cell Signaling Technologies, Beverly, MA, USA, Cat:#7074S). β-actin (Cell Signaling Technologies, Cat:#3700S) was the loading control. Goat anti-rabbit IgG (Cell Signaling Technologies, Cat:#7076S) and goat anti-mouse IgG were used to detect ANGPTL2 and β-actin, respectively.

All cell lines were obtained from Cell Resource Center of the Institute of Basic Medicine Sciences Chinese Academy of Medical Sciences (IBMS & CAMS, Beijing, China). HEK293 cells were washed once with cold PBS and solubilized on ice in CelLytic M lysis buffer (C2978; Sigma-Aldrich) supplemented with protease inhibitors (100×, B14001; Bimake). Cleared lysates were harvested by centrifugation at 13,000g for 15 minutes at 4°C, and 1.0 mg of each lysate was used for immunoprecipitation. The lysates were incubated with EZview Red Anti-FLAG Agarose Beads Affinity Gel (F2426; Sigma-Aldrich) or EZview Red Anti-c-Myc Agarose Beads Affinity Gel (E6654; Sigma-Aldrich) and isotype control IgG antibody at 4°C with gentle rotation for 4 hours. After 3 washes with PBS, the immunoprecipitated proteins were recovered from the beads by boiling for 10 minutes in sample buffer. Rabbit anti-EAAT2 antibody (22515-1-AP; Proteintech) and rabbit anti-AMFR antibody (16675-1-AP; Proteintech) were used for in vivo coimmunoprecipitation from mouse hippocampal tissues, and immunoprecipitated proteins were detected with mouse anti-EAAT2 antibody (sc-365634; Santa Cruz Biotechnology) and mouse anti-AMFR antibody (sc-166358; Santa Cruz Biotechnology).

Cells (1 × 10⁶ cells per plate) were inoculated in 100 mm polystyrene plates and grown. At 70% confluence, cells were exposed to 10 and 20 µM TA for 24 h along with the untreated control. The cells (both dead and live) were pelleted, lysed, and processed for protein profiling studies^{72 –77 (link)}. After TA treatment, cell lysates were prepared using CelLytic M lysis buffer (sigma Aldrich, St. Louis, MO, USA). The whole cell lysates were subjected to SDS-PAGE and transfer process. The transferred membranes were probed with a rabbit anti- AceCS1 (D19C6) (#3658), anti- Phospho-Acetyl-CoA Carboxylase (Ser79) (D7D11) (#11818), anti- Acetyl-CoA Carboxylase (C83B10) (#3676), anti ATP-Citrate Lyase (#4332), anti- Phospho-ATP-Citrate Lyase (Ser455) (#4331), and anti-Lipin 1 (D2W9G) (#14906), anti-ACSL1 (D2H5) (#9189) (cell signaling, Danvers, MA, USA) trailed by a peroxidase-conjugated, anti-rabbit IgG antibody (cell signaling, Danvers, MA). Protein expression were then developed using ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA)^{78 (link)}.

Cells were washed with ice-cold PBS, harvested by scraping and then homogenized in CelLytic M lysis buffer (Sigma, Merck) supplemented with phosphatase and protease inhibitors (PhosSTOP and cOmplete, Roche). Lysates were incubated (1 hour, 4°C) and then centrifuged at 14,000 × g (10 minutes, 4°C). Protein samples were quantified using the Pierce BCA Protein Assay (Thermo Fisher Scientific), resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Merck). Membranes were blocked with Tris-buffered saline/0.5% Tween 20 (TBST, pH 7.5) containing 5% skim milk powder and probed overnight with primary antibodies against the following targets: PSMA (1H8H5, Thermo Fisher Scientific), EGFR (D38B1), HER2 (44E7), HER3 (D22C5), p-AKT(S473) (587F11), p-AKT (T308) (244F9), AKT (D38B1), p-S6 (S235/236) (D57.2.2E), Calnexin (C5C9) and Tubulin (9F3) were from Cell Signaling Technology, AR (Santa Cruz Biotechnology), GAPDH (GT239, GeneTex), and mGluR1 (07-617, Merck). Following incubation with horseradish peroxidase–linked secondary antibodies (Dako, Agilent Technologies), immunoreactive bands were visualized with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).

HEK293 and HEK293 cells stably expressing NOX5β were cultured as described above, then lysed with Cellytic M lysis buffer (Sigma-Aldrich) containing protease inhibitor (EDTA-free Protease Inhibitor Cocktail mini tablet, Sigma-Aldrich). Protein concentrations were determined by DC Protein Assay kit (Bio-Rad) per manufacturer’s instructions and 500 μg of protein was mixed with 2 μL mouse anti-β-actin primary antibody (Sigma-Aldrich) or 8 μL rabbit anti-NOX5 antibody (Proteintech), and PBS containing protease inhibitor (EDTA-free Protease Inhibitor Cocktail mini tablet, Sigma-Aldrich) for a total volume of 300 μL. Samples were incubated at 4°C while rotating for 2–3 h. After rotation, samples were added to a tube containing 50 μL of pre-washed Protein G Mag Sepharose beads (Cytiva) for the anti-β-actin antibody or Protein A Sepharose beads (Thermo Scientific) for the anti-NOX5 antibody. The sample and bead mixture was incubated at 4°C while rotating for 16 h. After incubation the supernatant was removed using a magnetic rack (Protein G Mag Sepharose beads) or centrifugation (Protein A Sepharose beads) and the beads washed 3 times with PBS (Sigma-Aldrich). 30 μL of 2x sample buffer (Bio-Rad) was added to each sample, boiled at 100°C for 5 min and subjected to SDS-page and western blot analysis as described above.