Fusion software

Glycated proteins were identified as previously described13 (link). Briefly, brain homogenates were incubated with 0.5 mM fluorescent boronic acid at room temperature for one hour. Gel electrophoresis was performed using an Xcell surelock mini-cell (Invitrogen), separating proteins on 15% tris-glycine gels. Glycation was visualised prior to protein staining with a Dark Reader^® (Clare Chemicals Research Inc.; 420–520 nm, with 530 or 595 nm filter). Gels were then stained with coomassie to visualise total protein content. Samples where only SDS-PAGE was required skipped the boronic acid incubation and visualisation stages. Quantification of protein band fluorescence and coomassie stain intensity was performed using Fusion software (Vilber Lourmat).

The tissue samples and cells were lysed using RIPA Lysis Buffer (Beyotime Biotech, Shanghai, China) containing PMSF (Beyotime Biotech, Shanghai, China). The protein concentration was detected using the BCA kits. Equal amounts of protein were separated by 10%SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After blocking with 5% nonfat milk in Tris-buffered saline-Tween (TBST) for 2h at room temperature, membranes were incubated with primary antibodies against ORAI1(1:500, Proteintech, Wuhan, China), E-cadherin (1:1000, Santa Cruz, CA, USA), N-cadherin (1:1000, Cell Signaling Technology, MA, USA)), Vimentin (1:1000, Santa Cruz, CA, USA), β-actin (1:2000, Proteintech, Wuhan, China) overnight at 4°C. After washing with TBST, membranes were incubated with HRP-labeled secondary antibodies for 2h at room temperature. Protein bands were detected using an enhanced chemiluminescence detection kit (ECL). Densitometric analysis was performed using Fusion software (Vilber Lourmat, France). β-actin was used as loading control.

iPS cells and neurospheres were isolated, suspended in RIPA lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich), triturated and centrifuged at 10,000g, and 4°C for 10 min. The supernatants were separated on 10% SDS–PAGE gels (10 μg protein/lane) and blotted onto a polyvinylidene difluoride membrane. The membranes were then blocked with 5% skim milk, incubated with anti-AGE antibodies (CML: TransGenic, KH001, dilution 1/500; CEL: COSMO BIO, AGE-M02, dilution 1/1,000; MG-H1: CELL BIOLABS, STA-011, dilution 1/1,000; PEN: TransGenic, KH012, dilution 1/500; ARP: NOF CORPORATION, 5F6, dilution 1/500), or anti-GAPDH antibody (sc-20357, dilution 1/1,000; Santa Cruz Biotechnology) at 4°C overnight and then incubated with HRP-conjugated IgG (dilution 1/5,000; GE Healthcare) for 1 h at room temperature. The resulting signals were detected with an Immobilon Western Chemiluminescent HRP Substrate (Merck MilliporeSigma), and the bands were analyzed using a FUSION Solo instrument (Vilber Lourmat). The intensities of the bands were quantified using Fusion software (Vilber Lourmat). The expression level of GLO1 was normalized to that of GAPDH.

Schematic illustration of the experiments is shown in Fig. 3d. For determining t_1/2^surface (path-1 in Fig. 3d), first step labeling was conducted as described in “Two-step labeling of AMPARs or NMDARs in HEK293T cells.” After medium exchange for removal of the labeling reagents, the cells were incubated for 0, 1,4, 6, 12, 24, and 36 h. The cells were treated with 1 µM Tz(Fl) in PBS for 5 min, and excess Tz(Fl) was quenched by addition of 1 µM TCO-PEG4-COOH in PBS.
For determining t_1/2^life (path-2 in Fig. 3d), after first step labeling, cells were washed three times with culture medium and treated with 1 µM Tz(Fl) in culture medium for 5 min. Excess Tz(Fl) was quenched by addition of 1 µM TCO-PEG4-COOH in culture medium. Cells were then incubated for 0, 1,4, 6, 12, 24, and 36 h and washed three times with PBS. Cell lysis and western blotting were performed as described in “Two-step labeling of AMPARs or NMDARs in HEK293T cells.”
The target bands were manually selected, and the intensity were calculated with Fusion software (Vilber Lourmat), background intensity was manually subtracted by cutting the minimal intensity in the selected area. The half-life was calculated by curve fitting using KaleidaGraph and following equation (3): I = a + b·e^−ct, and the offset value (a) was set equal to zero. The t_1/2 was defined as t_1/2 = ln(2)/c.