anti pmtor apc  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a KEGG pathway enrichment of DEGs in ZIKV-infected U251 cells transfected with HA-DISC1 (1 μg) plasmid compared with the control group transfected with pcDNA. b KEGG pathway enrichment of differentially expressed proteins identified by IP/MS analysis in U251 cells overexpressing HA-DISC1 (5 μg) plasmid compared with the control group transfected with pcDNA. c Chord diagram illustrates the proteins identified in the IP/MS analysis. d U251 cells were transfected with pcDNA or HA-DISC1 (1 μg) plasmids for 24 h followed by ZIKV infection. The protein levels of AMPKα, pAMPKα, mTOR, pmTOR, P62 and LC3A/B were assessed by Western blot at 0, 12, 24, and 36 h post-infection. e U251 cells were transfected with pcDNA or HA-DISC1 (1 μg) plasmids for 24 h, followed by ZIKV infection for 6 h and treated with DMSO, rapamycin (25 μM), and MHY1485 (10 μM) for 24 h. ZIKV E expression was assessed by Western blot. f U251 cells were transfected with pcDNA or HA-DISC1 (1 μg) plasmids for 24 h, followed by ZIKV infection for 6 h and treated with DMSO, autophagosome inhibitor 3-MA (1 mg/ml), and lysosome inhibitor CQ (100 μM) for 24 h. ZIKV E expression was assessed by Western blot. KEGG pathway enrichment analyses in ( a , b ) were performed using a one-tailed Fisher’s exact test. Images in ( d − f ) are one representative experiment out of three independent replicates with similar results ( n = 3). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-DISC1 antibody (1:1000, Invitrogen, 40-6800), anti-DISC1 antibody (1:500, Santa Cruz Biotechnology, sc-365591), anti-DISC1 antibody (1:500, GeneTex, GTX33154), anti-Zika virus E antibody (1:000, Genetex, GTX133314), anti-HA tag antibody (1:2000, Genetex, GTX115044), anti-AMPKα antibody (1:1000, Beyotime, AF1627), anti-pAMPKα antibody (1:1000, Cell Signaling Technology, 2535S), anti-mTOR antibody (1:500, Beyotime, AF1648), anti-pmTOR antibody (1:1000, Cell Signaling Technology, 5536), anti-P62 antibody (1:1000, Cell Signaling Technology, 39749), anti-LC3A/B antibody (1:1000, Cell Signaling Technology, 4108), anti-GFP antibody (1:2000, Genetex, GTX113617), anti-Flag antibody (1:2000, Sigma, F1804), anti-DYNC1I2 antibody (1:1000, Proteintech, 12219-1-AP), anti-LIS1 antibody (1:1000, Proteintech, 20678-1-AP), anti-NDEL1 antibody (1:1000, Proteintech, 17262-1-AP), anti-GAPDH antibody (1:20000, Fude Biotech, FD0063), HRP conjugated goat anti-mouse antibody (1:10000, Fude Biotech, FDM007), HRP conjugated goat anti-rabbit antibody (1:10000, Fude Biotech, FDR007).

Techniques: Infection, Transfection, Plasmid Preparation, Control, Protein-Protein interactions, Western Blot, Expressing, One-tailed Test

Journal: Nature Communications

Article Title: DISC1 Protects Against Zika Virus Infection and Long-Term Neurological Damage Through AMPK-mTOR-Mediated Autophagy

doi: 10.1038/s41467-025-64809-w

Figure Lengend Snippet: a Primary cortical neurons were isolated from neonatal mice (P0–P1) and induced to differentiate in vitro. On day 7 of differentiation, neurons exhibiting neurite outgrowth were observed under bright-field microscopy. Confocal imaging showed neuronal nuclei stained with DAPI (blue), and endogenous Neun labeled with an anti-Neun antibody (red). Scale bar = 50 μm. b , c WT and Disc1 KD primary cortical neurons were pre-incubated with 20 μg/mL MAR1-5A3 for 6 h, followed by infection with ZIKV at a MOI of 1. Viral mRNA levels and titers were measured on D1 and D2 post-infection using qRT-PCR ( b ) and plaque assay ( c ). d WT and Disc1 KD primary cortical neurons were pre-incubated with 20 μg/mL MAR1-5A3 for 6 h, followed by infection with ZIKV at a MOI of 1. The protein levels of ZIKV E protein, DISC1, AMPKα, pAMPKα, mTOR, pmTOR, P62 and LC3A/B were assessed by Western blot on D1 and D2 post-infection. e, f The ratios of pAMPKα to AMPKα ( e ) and pmTOR to mTOR ( f ) were determined by comparing the corresponding protein band intensities at each time point in Fig. 8d. g − i Quantification of P62 ( g ), LC3A/BⅠ ( h ) and LC3A/BⅡ ( i ) protein levels relative to GAPDH at each time point in Fig. 8d. j , k Placentas ( j ) and fetal heads ( k ) from non-infected and ZIKV-infected WT and Disc1 KD mice were collected on E13.5. The protein levels of DISC1, AMPKα, pAMPKα, mTOR, pmTOR, P62 and LC3A/B were assessed by Western blot. l − o The ratios of pAMPKα to AMPKα ( l, m ) and pmTOR to mTOR ( n , o ) were determined by comparing the corresponding protein band intensities at each time point in Fig. 8j ( l, n ) and Fig. 8k ( m , o ). p − s Quantification of P62 ( p , q ) and LC3A/BⅡ ( r , s ) protein levels relative to GAPDH at each time point in Fig. 8j ( p , r ) and Fig. 8k ( q , s ). Data shown in ( b , c ) are from one representative experiment out of three independent replicates with similar results, each including 3 biological replicates per group ( n = 3). Images in ( a , d , j – k ) are from one representative experiment out of three independent replicates with similar results ( n = 3). Data shown in ( e − i , l − s) are collected from three independent experiments ( n = 3). Data shown in ( b − c , e − i , l − s ) were confirmed to follow a normal distribution using the Shapiro-Wilk test, and statistical analysis was performed using Student’s unpaired two-tailed t test ( b , c ) or two-tailed one-way ANOVA followed by Tukey’s multiple comparison test ( e − i , l − s ). Data are presented as means ± SD. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-DISC1 antibody (1:1000, Invitrogen, 40-6800), anti-DISC1 antibody (1:500, Santa Cruz Biotechnology, sc-365591), anti-DISC1 antibody (1:500, GeneTex, GTX33154), anti-Zika virus E antibody (1:000, Genetex, GTX133314), anti-HA tag antibody (1:2000, Genetex, GTX115044), anti-AMPKα antibody (1:1000, Beyotime, AF1627), anti-pAMPKα antibody (1:1000, Cell Signaling Technology, 2535S), anti-mTOR antibody (1:500, Beyotime, AF1648), anti-pmTOR antibody (1:1000, Cell Signaling Technology, 5536), anti-P62 antibody (1:1000, Cell Signaling Technology, 39749), anti-LC3A/B antibody (1:1000, Cell Signaling Technology, 4108), anti-GFP antibody (1:2000, Genetex, GTX113617), anti-Flag antibody (1:2000, Sigma, F1804), anti-DYNC1I2 antibody (1:1000, Proteintech, 12219-1-AP), anti-LIS1 antibody (1:1000, Proteintech, 20678-1-AP), anti-NDEL1 antibody (1:1000, Proteintech, 17262-1-AP), anti-GAPDH antibody (1:20000, Fude Biotech, FD0063), HRP conjugated goat anti-mouse antibody (1:10000, Fude Biotech, FDM007), HRP conjugated goat anti-rabbit antibody (1:10000, Fude Biotech, FDR007).

Techniques: Isolation, In Vitro, Microscopy, Imaging, Staining, Labeling, Incubation, Infection, Quantitative RT-PCR, Plaque Assay, Western Blot, Two Tailed Test, Comparison