Multi gauge 3

Western blot was performed as previously described^{16 (link)}. Cells were washed with PBS and lysed in Cytobuster Protein Extraction Reagent (Novagen Inc., Madison, Wisconsin, USA) supplemented with a protease inhibitor cocktail (Nacalai Tesque). Protein samples were separated on polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with the following primary antibodies: anti-CD9 (1:1000, Ts9, Thermo Fisher Scientific), anti-NT5E/CD73 (1:1000, D7F9A, Cell Signaling Technology), anti-CD63 (1:1000, Ts63, Thermo Fisher Scientific), anti-CD81 (1:1000, Ts81, Thermo Fisher Scientific), anti-HIF-1α (1:1000, D1S7W, Cell Signaling Technology), anti-mTOR (1:1000, 7C10, Cell Signaling Technology), anti-p-mTOR (1:1000, S2448, Cell Signaling Technology), anti-CD5L/CD-2 (1:1000, ab45408, Abcam), and anti-β-actin (1:1000, 13E5, Cell Signaling Technology). Blotted membranes were visualized using a densitometry technique on a LAS-4000 mini luminescent image analyzer (GE Healthcare, Marlborough, MA, USA) and quantified using Multi Gauge 3.1 software (FUJIFILM, Tokyo, Japan). For reference, all full-length blots are presented in Supplementary Materials.

Western blotting was performed as previously described (Watanabe et al., 2022 (link)). Cells were washed with phosphate-buffered saline and lysed in Passive Lysis 5× buffer (Promega, Madison, WI, United States). Nuclear proteins were extracted using a LysoPure Nuclear and Cytoplasmic Extractor Kit (FUJIFILM, Tokyo, Japan). Protein samples were separated on polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with the appropriate primary antibodies, anti-ATF6β (1:1000; 15794-1-AP, Proteintech, Rosemont, IL, United States), anti-RANKL (1:1000; 12A668, Novus Biologicals, United States), anti-CHOP (1:1000, L63F7, Cell Signaling Technology), anti-Grp78 (1:1000, C50B12, Cell Signaling Technology), anti-cleaved caspase-3 (1:1000, 5A1E, Cell Signaling Technology), anti-JNK (1:1000, 56G8, Cell Signaling Technology), anti-Phospho-JNK (Thr183/Tyr185) (1:1000, 81E11, Cell Signaling Technology), anti-Lamin B1 (1:1000, 1298-1-AP, Proteintech) and anti-β-actin (1:1000; 13E5, Cell Signaling Technology) antibodies, and secondary antibodies, anti-rabbit IgG (1:2000; Cell Signaling Technology) and anti-mouse IgG (1:2000; Cell Signaling Technology). Blotted membranes were visualized using a densitometry technique on Amersham ImageQuant 800 (Cytiva, Tokyo, Japan) and quantified using Multi Gauge 3.1 software (FUJIFILM, Tokyo, Japan).

Western blotting was performed as described previously [14 (link)]. The cells were washed with PBS, lysed in Passive Lysis 5x buffer (Promega, Madison, WI, USA), and supplemented with a protease inhibitor cocktail (Nacalai Tesque). Protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan). The membranes were incubated with Blocking One-P (Nacalai Tesque) and probed with the following primary antibodies: anti-GAPDH (14C10) (1 : 1000, #2118; Cell Signaling Technology, Danvers, MA), anti-PKR (B-10) (1 : 1000, sc-6282; Santa Cruz Biotechnology, Dallas, TX), and anti-PKR (phospho T446) [E120] (1 : 200, ab32036; Abcam). Blotted membranes were detected using Chemi-Lumi One Super (Nacalai Tesque) and visualized using densitometry on an Amersham ImageQuant 800 platform (Cytiva, Tokyo, Japan). Immunoblots were quantified using Multi Gauge 3.1 software (FUJIFILM, Tokyo, Japan).

Enzymes were extracted from frozen leaf material (150 mg FW) that had been stored at –80°C. GS activity was determined using the γ-glutamyl transferase method, and GS western blots for the GS1 and GS2 proteins were prepared according to Lothier et al. (2011) (link). Antibodies raised against the GS1 and GS2 isoenzymes were produced by Lemaître et al. (2008) (link). Quantification of signals on the western blot membranes was performed using the Multi Gauge 3.2 software (Fujifilm).
The relative proportions of GS activity due to the chloroplastic and cytosolic isoforms were evaluated after fractionation. Plant material was extracted (2 g FW in 10 ml extraction buffer) according to Lothier et al. (2011) (link). After centrifugation (15 000 g 15 min at 4°C), the supernatant was filtered (0.2-µm filter, GelmanSciences) and injected into a Mono Q anion exchange column (5/50 GL, GE Healthcare) attached to a FPLC system (ÄKTApurifier, GE Healthcare). The Mono Q column had been pre-equilibrated with 30 ml of equilibration buffer (25 mM Tris-HCl, 1 mM MgCl₂, 1 mM EDTA) before loading. FPLC was performed at room temperature. Protein fractions were eluted from the column using a linear gradient from 0.1 to 0.7 M NaCl with a flow rate of 1.0 ml min^–1. Fifty fractions (500 µl) were collected and assayed for GS activity. Aliquots were denatured for SDS-PAGE and western blotting assays.

Immediately after microPET imaging, the rats were euthanized and the stomachs excised along the lesser curvature (Fig 1F). Excised stomachs were transferred to a chilled autoradiography cassette and stored for 12 h at -4°C. Screens were read using an FLA7000 scanner (Fujifilm, Tokyo, Japan). ROIs were selected on the greater curvature of the stomach where the blood supply was intact, and in the fundus where the blood supply was not intact because of partial devascularization of the stomach. The optical densities of autoradiographic signals were measured using Multi Gauge 3.2 software (Fujifilm, Tokyo, Japan). Autoradiographic images and ROIs were compared between the two groups.