Journal: Nature

Article Title: A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol

doi: 10.1038/s41586-020-2076-4

Figure Lengend Snippet: a, 293T cells were co-transfected with StrepTagII-HA-HRI and the indicated Flag-mNeon-tagged cDNAs before stimulation with CCCP as indicated. Cells were then lysed and StrepTagII-containing protein complexes were immobilized on Strep-Tactin beads and analysed by immunoblotting along with the input lysate. DELE1FL, full-length DELE1. b, A C-terminal in-frame fusion of a triple Flag tag was introduced into the endogenous EIF2AK1 (HRI) locus of 293T cells using CRISPR. c, Quantification of Fig. 3b. The bar chart shows the relative abundance of the slowly migrating species of HRI (phosphorylated HRI) compared to the faster migrating band. Data were normalized to the empty vector (black) and significance (compared to full-length DELE1 untreated, grey) was assessed using one-way ANOVA with Dunnett’s multiple comparisons correction (mean ± s.d. of n = 3 independent experiments). d, 293T cells were co-transfected with HRI-Flag and DELE1-HA and exposed to CCCP for the denoted amounts of time before immunoblotting. The shift in electrophoretic mobility of HRI upon activation is reversed by treatment of the lysates with alkaline phosphatase. e, 293T cells of the indicated genotypes were co-transfected with HRI-Flag and the specified HA-tagged constructs. After CCCP treatment, lysates were immobilized on anti-Flag beads and analysed by immunoblotting along with the input lysate (one representative experiment shown of four independent experiments). f, Quantification of e(mean ± s.d. of n = 4 independent experiments; two-way ANOVA with Tukey’s multiple comparisons correction). g, HeLa cells were transiently transfected with the indicated constructs, treated with CCCP or DMSO and analysed for subcellular localization of the transfected cDNAs by HA staining and confocal microscopy with cellular structures labelled as indicated. h, DELE1 mutants were transiently transfected into HeLa and 293T cells before exposure to CCCP for 2 h. The processing of DELE1–HA was monitored by immunoblotting. i, HeLa cells were transfected as indicated and analysed for subcellular localization as in g. j, 293T cells were co-transfected with HRI-Flag and the indicated StrepTagII-HA cDNAs. Cells were then lysed and StrepTagII-containing protein complexes were immobilized on Strep-Tactin beads and analysed by immunoblotting along with the input lysate. k, l, Clonal DELE1-knockout HAP1 cells were transiently transfected with the specified cDNAs together with mCherry and induction of CHOPNeon was monitored by flow cytometry as in Fig. 2a. Per genotype, data were normalized to the empty vector and statistical significance (compared to DELE1(ΔMTS(N101))) was assessed using one-way ANOVA with Tukey’s multiple comparisons correction (mean ± s.d. of n = 4 independent experiments). m, Wild-type 293T cells were transiently transfected with HRI-Flag and the indicated HA-tagged cDNAs and processed as in e (one representative experiment shown of four independent experiments). n, DELE1-deficient HAP1 CHOPNeon cells were transiently transfected as indicated and analysed by flow cytometry 32 h after transfection. Per genotype, data were normalized to the empty vector (black) and statistical significance (compared to DELE(ΔMTS)) was assessed using one-way ANOVA with Tukey’s multiple comparisons correction (mean ± s.d. of n = 3 independent experiments). o, Cell lysates from Fig. 3c were analysed by Coomassie staining before (lysate) and after exposure to GFP-TRAP beads (IP (GFP)). Scale bars, 10 μm (g, i).

Article Snippet: In the latter case, DELE1(TPR1–7)–EGFP and EGFP were detected using a rat anti-GFP antibody (Chromotek, 3h9) different from the alpaca nanobody conjugated to the GFP-TRAP beads, whereas GST–HRI was detected using an antibody directed against GST (GE Healthcare, 27457701V).

Techniques: Transfection, Western Blot, FLAG-tag, CRISPR, Plasmid Preparation, Activation Assay, Construct, Staining, Confocal Microscopy, Knock-Out, Flow Cytometry

Journal: Nature

Article Title: A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol

doi: 10.1038/s41586-020-2076-4

Figure Lengend Snippet: a, Schematic depiction of haem metabolism. b, Data from Fig. 1a, with statistical significance of mutation enrichment assessed and visualized in the same way (two-sided Fisher’s exact test, P adj < 0.05). Haem-related genes from a are highlighted in red. c, Immunoprecipitation experiment as in Extended Data Fig. 9a in the presence of haemin during cell culture (20 μM for 12 h before lysis) and immunoprecipitation (8 μM in lysis and wash buffers). HMOX1 is induced in response to increased haem levels (one representative experiment shown of two independent experiments). d, Model of the pathway identified here. Mitochondrial stress activates OMA1, which leads to cleavage of DELE1 and its subsequent cytosolic binding to and activation of HRI, triggering the ISR. e, Wild-type HAP1 and HeLa cells were treated with CCCP, and the mitochondrial membrane potential was visualized by TMRM using a live-cell-imaging system. H2B-GFP was transfected to stain nuclei. Scale bars, 100 μm. f, g, HAP1 CHOPNeon cells were stimulated as indicated, and CHOPNeon levels (g) and the mitochondrial membrane potential (f) were quantified by flow cytometry. Statistical significance is depicted relative to DMSO-treated control cells (mean ± s.d. of n = 3 independent experiments; one-way ANOVA with Tukey’s multiple comparisons correction). h, HeLa cells were transfected with the indicated sgRNAs and the mitochondrial membrane potential was quantified after 1 h of CCCP treatment by flow cytometry. The TMRM signal was normalized to the non-targeting control sgRNA and DMSO control, and for CCCP-treated samples statistical significance was assessed relative to the non-targeting control sgRNA and CCCP (mean ± s.d. of n = 3 independent experiments; two-way ANOVA with Dunnett’s multiple comparisons correction). i, Volcano plots of RNA-seq analysis of the indicated samples. For every gene, the fold change in expression after CCCP treatment (versus DMSO treatment; x axis) is plotted against the P value (two-tailed Wald test in DESeq2; y axis). Genes with a significant differential expression (P adj < 10−12 (Benjamini–Hochberg)) are depicted in dark grey. ATF4 and CHOP target genes39,40 (green) and heat-shock proteins43 (red) from Fig. 3e are highlighted (n = 4 biologically independent samples per group). FAM129A is also known as NIBAN1.

Article Snippet: In the latter case, DELE1(TPR1–7)–EGFP and EGFP were detected using a rat anti-GFP antibody (Chromotek, 3h9) different from the alpaca nanobody conjugated to the GFP-TRAP beads, whereas GST–HRI was detected using an antibody directed against GST (GE Healthcare, 27457701V).

Techniques: Mutagenesis, Immunoprecipitation, Cell Culture, Lysis, Binding Assay, Activation Assay, Live Cell Imaging, Transfection, Staining, Flow Cytometry, RNA Sequencing Assay, Expressing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Aurora A kinase phosphorylation of Hec1 S69 is required for metaphase kinetochore function. (A) Kymographs of individual sister kinetochore pairs from live-cell time-lapse imaging sequences of PtK1 cells depleted of endogenous Hec1 and rescued with WT-Hec1-GFP (WT) or mutants of Hec1 with a single phosphorylation site mutated to alanine: S69A, S55A, and S44A. Cells were treated with 10 µM MG132 before imaging. Quantification of the deviation from average position is shown on the right. For each condition, ≥30 kinetochore pairs from a total of nine cells were analyzed. (B) Immunofluorescence images of untreated PtK1 cells or cells treated with 1 µM MLN8054 (MLN) or 2 µM ZM447439 (ZM). All cells were additionally treated with 10 µM MG132 at the time of kinase inhibitor addition. After 1 h, cells were fixed and stained with antibodies to phosphorylated S69 (pS69). Bar, 10 µm. Shown on the right is the quantification of pS69 kinetochore fluorescence intensity. For each condition, ≥200 kinetochores were measured from ≥20 cells. (C) Kymographs of individual sister kinetochore pairs from live-cell time-lapse imaging sequences of untreated PtK1 cells or cells treated with 1 µM MLN8054, 2 µM ZM447439, or both inhibitors. All cells were treated with 10 µM MG132 at the time of inhibitor addition. Quantification of the deviation from average position is shown on the right. For each condition, ≥20 kinetochore pairs were measured from at least five cells. (D) Kymographs of individual sister kinetochore pairs from live-cell time-lapse imaging sequences of PtK1 cells depleted of endogenous Hec1 and rescued with WT-Hec1-GFP or mutants of Hec1 with either S69 or S55 mutated to aspartic acid and the eight remaining phosphorylation target sites mutated to alanine (e.g., 8A-S69D-Hec1-GFP). Cells were treated with 1 µM MLN8054 and 10 µM MG132 before imaging. Quantification of the deviation from average position is shown on the right. For each condition, ≥20 kinetochore pairs were measured from at least five cells. (E) Kymographs of individual sister kinetochore pairs from live-cell time-lapse imaging sequences of PtK1 cells depleted of endogenous Hec1 and rescued with WT-Hec1-GFP, 9A-Hec1-GFP, or mutants of Hec1 with all phosphorylation target sites except for one (S69, S55, or S44) mutated to alanine. The remaining site was left as an unperturbed serine (e.g., 8A-S69 WT -Hec1-GFP). Quantification of the deviation from average position is shown on the right. For each condition, ≥20 kinetochore pairs were measured from at least five cells. For all kymographs, distance scale bars are 1 µm, and time scale bars are 15 s. Error bars indicate SD. All kymographs represent individual sister kinetochore pairs displayed over time.

Article Snippet: The following primary antibody dilutions were used: mouse anti-Hec1 9G3 at 1:3,000 (Novus Biologicals), human anticentromere antibody at 1:300 (Antibodies, Inc.), DM1α mouse antitubulin at 1:200 (Sigma-Aldrich), rabbit antiphosphorylated Aurora A (pT288) at 1:500 (Cell Signaling Technology), rabbit antiphosphorylated Aurora B (pT232) at 1:1,000, rabbit antiphosphorylated Hec1 Ser44 (pS44) at 1:3,000, rabbit antiphosphorylated Hec1 Ser55 (pS55) at 1:1,000, rabbit antiphosphorylated Hec1 Ser69 (pS69) at 1:3,000, mouse anti-INCENP at 1:1,000 (Abgent), rat anti-GFP at 1:1,000 (ChromoTek), mouse anti–Aurora A at 1:500 (BD), and guinea pig anti–Cenp-C at 1:1,000 (MBL).

Techniques: Imaging, Immunofluorescence, Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Inhibition of Hec1 S69 phosphorylation results in chromosome segregation errors and altered mitotic timing. (A) HeLa Flp-In T-REx cells depleted of endogenous Hec1 and induced to express either WT- or S69A-Hec1-GFP were fixed and scored for segregation errors. Errors included for quantification were lagging anaphase chromosomes and anaphase bridges. Examples of WT- and S69A-Hec1-GFP–expressing cells in anaphase are shown (chromosomes stained with DAPI are in red, and Hec1-GFP is in green). The arrow points to lagging chromosomes; this region is shown magnified to the right. For the quantification, ≥400 cells from four experiments were analyzed. Error bars represent SD. Bars: (main image) 10 µm; (magnified image) 1 µm. (B) Quantification of mitotic timing of HeLa Flp-In T-REx cells depleted of endogenous Hec1 and induced to express either WT- or S69A-Hec1-GFP. Mitotic progression was scored from nuclear envelope breakdown (NEB) to metaphase (M) and from metaphase to anaphase onset (AO). Total time in mitosis was scored from nuclear envelope breakdown to anaphase onset. For each condition, ≥75 cells were analyzed.

Article Snippet: The following primary antibody dilutions were used: mouse anti-Hec1 9G3 at 1:3,000 (Novus Biologicals), human anticentromere antibody at 1:300 (Antibodies, Inc.), DM1α mouse antitubulin at 1:200 (Sigma-Aldrich), rabbit antiphosphorylated Aurora A (pT288) at 1:500 (Cell Signaling Technology), rabbit antiphosphorylated Aurora B (pT232) at 1:1,000, rabbit antiphosphorylated Hec1 Ser44 (pS44) at 1:3,000, rabbit antiphosphorylated Hec1 Ser55 (pS55) at 1:1,000, rabbit antiphosphorylated Hec1 Ser69 (pS69) at 1:3,000, mouse anti-INCENP at 1:1,000 (Abgent), rat anti-GFP at 1:1,000 (ChromoTek), mouse anti–Aurora A at 1:500 (BD), and guinea pig anti–Cenp-C at 1:1,000 (MBL).

Techniques: Inhibition, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Aurora A kinase binds and localizes to INCENP. (A) Volcano plot of quantitative liquid chromatography–tandem mass spectrometry experiments identifying interactors of INCENP. Plotted are the differences in label-free quantification intensity between the INCENP-GFP (right) and control group against the transformed (−log 10 ) p-value of a Student’s t test. The red line indicates the permutation-based false discovery rate (FDR) threshold (0.001) to correct for multiple testing. (B) Western blot analysis of an anti-GFP immunoprecipitation (IP) performed in mitotic cell lysates, using either nocodazole (N) or STLC (S) from HeLa cells expressing either LAP or INCENP-LAP. After SDS-PAGE, the Western blot was probed for GFP and Aurora A kinase (AAK). 10% of input was loaded. (C) Western blot analysis of an anti-GFP immunoprecipitation performed in cell lysates from HEK-293T cells transiently expressing either GFP, full-length (FL) INCENP-GFP, or various truncation mutants of INCENP-GFP (1–820, 1–878, and 1–897). The Western blot was probed for GFP and Aurora A kinase and reprobed for Aurora B kinase (ABK). 10% of input was loaded. (D) Immunofluorescence images of U2OS cells expressing either LacI-mCherry or LacI-mCherry-ΔCEN-INCENP (a version of INCENP lacking the N-terminal 57 residues; ). Cells were immunostained with an Aurora A kinase antibody. Enlarged images show recruitment of Aurora A kinase to the LacI-mCherry-ΔCEN-INCENP spot. Quantification of Aurora A kinase intensity is shown on the right. (E) Immunofluorescence images of U2OS cells expressing VSV-INCENP-GFP under a doxycycline-inducible promoter, either noninduced or induced. Cells were immunostained with GFP and Aurora A kinase antibodies. Enlarged images show colocalization of INCENP-GFP and Aurora A kinase upon overexpression of INCENP-GFP. (F) Line graphs of kinetochore pairs highlighted in E. (G) Immunofluorescence images showing kinetochore localization of pS69 in HeLa cells treated with siRNA to either luciferase (control) or INCENP (siINCENP). Quantification of pS69 and INCENP fluorescence intensity is shown on the right. For pS69 quantification, n = 3 experiments of 15–20 cells; for INCENP quantification, n = 1 experiment of 15–20 cells. Error bars indicate SD. ****, P < 0.0001; unpaired t test. Bars: (main images) 10 µm; (insets) 1 µm.

Article Snippet: The following primary antibody dilutions were used: mouse anti-Hec1 9G3 at 1:3,000 (Novus Biologicals), human anticentromere antibody at 1:300 (Antibodies, Inc.), DM1α mouse antitubulin at 1:200 (Sigma-Aldrich), rabbit antiphosphorylated Aurora A (pT288) at 1:500 (Cell Signaling Technology), rabbit antiphosphorylated Aurora B (pT232) at 1:1,000, rabbit antiphosphorylated Hec1 Ser44 (pS44) at 1:3,000, rabbit antiphosphorylated Hec1 Ser55 (pS55) at 1:1,000, rabbit antiphosphorylated Hec1 Ser69 (pS69) at 1:3,000, mouse anti-INCENP at 1:1,000 (Abgent), rat anti-GFP at 1:1,000 (ChromoTek), mouse anti–Aurora A at 1:500 (BD), and guinea pig anti–Cenp-C at 1:1,000 (MBL).

Techniques: Liquid Chromatography, Mass Spectrometry, Transformation Assay, Western Blot, Immunoprecipitation, Expressing, SDS Page, Immunofluorescence, Over Expression, Luciferase, Fluorescence

Journal: eLife

Article Title: Peptidoglycan-dependent NF-κB activation in a small subset of brain octopaminergic neurons controls female oviposition

doi: 10.7554/eLife.50559

Figure Lengend Snippet:

Article Snippet: Rat anti-RFP [5F8] , ChromoTek Cat# 5f8-100, RRID: AB_2336064 , 1:1000.

Techniques:

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Experimental scheme for this study. After gene identification at 1 month of age, 3-month-old male WT and 3×Tg-AD mice were randomly assigned to four groups with 10 animals each and then intragastrically administered either ICA or vehicle for 5 months (WT + vehicle, WT + ICA, 3×Tg-AD + vehicle, 3×Tg-AD + ICA groups). After performing behavior tests, the mice were euthanized. The cerebral cortexes were evaluated using HE and Nissl staining, immunofluorescent staining, and western blot assays to determine the above disease indicators. 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; Aβ: beta-amyloid protein; AKT: protein kinase B; APP: amyloid precursor protein; GLUT: glucose transporter; GSK3β: glycogen synthase kinase 3 beta; HE: hematoxylin and eosin; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; NeuN: neuronal nuclear antigen; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PSD95: postsynaptic density protein 95; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Staining, Western Blot, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Effects of ICA on the pathological injury in the cerebral cortex of 3×Tg-AD mice. (A) Pathological changes in the cerebral cortex detected by HE and Nissl staining (original magnification 400×, scale bars: 50 μm). ICA treatment alleviated pathological changes of neurons in 3×Tg-AD mice, including irregular arrangements, structural ambiguities, nuclear shrinkage and deep staining, and markedly promoted neuronal survival. The arrows indicate injured neurons. (B) The numbers of surviving neurons detected by Nissl staining. (C) The numbers of NeuN-positive neurons (red, fluorescein) detected by immunofluorescent staining (original magnification 200×, scale bars: 200 μm). NeuN-positive neurons were significantly decreased in 3×Tg-AD mice, while ICA treatment of 3×Tg-AD mice observably increased the number of NeuN-positive neurons. (D) Representative expression patterns of NeuN and PSD95 in mouse cerebral cortexes detected by western blotting. (E) Quantification of NeuN and PSD95 protein expression. Expression levels were normalized to those of the WT + vehicle group. The data are presented as the means ± SEM ( n = 3–4). * P < 0.05, vs . WT + vehicle group; # P < 0.05, vs . 3×Tg-AD + vehicle group (one-way analysis of variance followed by the least significant difference test). 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; DAPI: 4′-6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HE: hematoxylin and eosin staining; ICA: icariin; Nissl: Nissl staining; NeuN: neuronal nuclear antigen; PSD95: postsynaptic density protein 95; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Staining, Expressing, Western Blot, Transgenic Assay