k85a gsk3β  (Addgene inc)

Journal: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: Wild-type MEFs were transfected with adenovirus encoding βGal (control), wild-type (WT) or kinase-active (K85R, DN) GSK3β for 48hrs. A ) Western blotting for GSK3β demonstrating equivalent expression of the WT-GSK3β and DN-GSK3β. GAPDH was used as a loading control. B ) βGal, WT-GSK3β and DN-GSK3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Transfection, Control, Western Blot, Expressing, Infection, Staining, Fluorescence

Journal: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: Wild-type MEFs were transfected with non-targeting (CONsi) or GSK3β-specific (GSK3βsi) siRNAs (100nM) for 48hrs. A ) Western blotting for GSK3β confirmed depletion of the protein in GSK3βsi-transfected MEFs. GAPDH was used as a loading control. B ) CONsi and GSK3βsi-transfected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of CRC measurements using Calcium Green-5N in digitonin-permeabilized CONsi and GSK3βsi-transfected MEFs. D ) Quantification of CRC data in the CONsi and GSK3βsi-transfected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. CONsi.

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Transfection, Western Blot, Control, Staining

Journal: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: A ) Upper panels, Western blotting for GSK3β, lactate dehydrogenase (LDH, cytosolic), and succinate dehydrogenase (SDHB, mitochondrial) in cardiac mitochondrial and cytosolic subfractions. Lower panels, Western blotting for GSK3β, VDAC (membrane), and CypD (soluble) in alkali extracted membrane and soluble fractions of cardiac mitochondria. B ) Upper panels, recombinant GST-rat GSK3β was incubated with His-tagged human CypD, purified on Co 2+ -agarose column, and the complexes Western blotted for GST and CypD. Lower panels, recombinant GST-GSK3β was incubated with His-tagged human CypD in kinase buffer supplemented with ATP, purified on Co 2+ -agarose column, and the complexes Western blotted for phosphoserine/threonine, GST and CypD. C ) Mass analysis of phosphorylated recombinant CypD demonstrating the presence of two phosphorylation sites. D ) Example of MS-MS spectra indicating S101 as a putative GSK3β phosphorylation site on CypD. E ) Amino acid sequence of the recombinant His-tagged human CypD depicting canonical GSK3β consensus sites (red), the putative phosphorylation sites identified in our analyses (blue), and a site previously hypothesized to be a key GSK3β phosphorylation residue (purple). F ) Surface representations of human CypD illustrating the positions of the various putative phosphorylation sites and their proximity to the catalytic CsA-binding domain (CsA-BD).

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Western Blot, Membrane, Recombinant, Incubation, Purification, Phospho-proteomics, Tandem Mass Spectroscopy, Sequencing, Residue, Binding Assay

Journal: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: Wild-type MEFs were infected with adenoviruses encoding βGal (control) or mitochondrially-targeted HA-tagged WT-GSK3β (miWT-3β) or DN-GSK3β (miDN-3β) for 48hrs. A ) Western blotting for HA and GSK3β confirmed equivalent expression of the two GSK3β proteins. GAPDH was used as a loading control. B ) Representative images of immunocytochemistry performed on MEFs infected with miWT-3β stained for the HA tag confirmed mitochondrial localization of the protein. Mitochondria were visualized by staining for CypD. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, miWT-3β and miDN-3β-infected MEFs. Cells were treated with 2.5μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, miWT-3β and miDN-3β-infected MEFs. E ) βGal, miWT-3β and miDN-3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. Each individual point represents one independent cell isolate. Bar represents the mean.

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Infection, Control, Western Blot, Expressing, Immunocytochemistry, Staining, Fluorescence

Journal: bioRxiv

Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

doi: 10.64898/2026.01.15.699680

Figure Lengend Snippet: A) Cardiac mitochondria were exposed to increasing concentrations of proteinase K and then Western blotted for hexokinase-2 (HK2, mitochondrial surface), apoptosis-inducing factor (intermembrane space), ubiquinol-cytochrome c reductase core protein 2 (UQCRC2, inner membrane), and CypD (matrix). B) MEFs were infected with adenoviruses encoding βGal, WT-GSK3β and/or FLAG-tagged CypD for 48hrs. CypD was immunoprecipitated using an anti-FLAG antibody and the complexes Western blotted for GSK3β and FLAG. C) CypD KO MEFs were infected with adenoviruses encoding βGal, WT-GSK3β for 48hrs. Western blotting for GSK3β confirmed overexpression of GSK3β. GAPDH was used as a loading control. D) βGal and WT-GSK3β-infected CypD KO MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. E) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, and WT-GSK3β -infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. F) Quantification of CRC data in the βGal and WT-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.

Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

Techniques: Western Blot, Membrane, Infection, Immunoprecipitation, Over Expression, Control, Staining, Fluorescence

Journal: Aging Cell

Article Title: Long‐term exposure to excessive norepinephrine in the brain induces tau aggregation, neuronal death, and cognitive deficits in early tau transgenic mice

doi: 10.1111/acel.14420

Figure Lengend Snippet: RBX facilitates the activities of tau kinases, PKA and GSK3β. (a, b) Representative western blot images. (c, d) Quantification of specific antibodies against pPKA, PKA, pGSK3β (S9), pGSK3β (Y216), or GSK3β in the hippocampus following treatments in the ADLP Tau mice. One dot in the bar graphs represents each mouse (c, d). All data represent mean ± SEM. Significance was determined by unpaired t test and denoted by * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001.

Article Snippet: The following day, the cells were transfected with plasmids encoding GSK3β WT (Addgene, #14753), GSK3β S9A (Addgene, #14754), or GSK3β K85A (Addgene, #14755) using Lipofectamine LTX (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Western Blot

Journal: Aging Cell

Article Title: Long‐term exposure to excessive norepinephrine in the brain induces tau aggregation, neuronal death, and cognitive deficits in early tau transgenic mice

doi: 10.1111/acel.14420

Figure Lengend Snippet: Human brain organoids exposed to the elevated NE facilitate the activities of PKA and GSK3β and aggravate tau aggregation. (a) Schematic representation of the experimental design for generating human brain organoids. (b–f) Representative western blot images and quantification of specific antibodies against 6E10, AT8, Tau13, pPKA, PKA, pGSK3β (Y216), or GSK3β in the human brain organoids exposed to various levels of NE. One dot in the bar graphs represents each organoid (b–f). All data represent mean ± SEM. Significance was determined by one‐way ANOVA and denoted by * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001.

Article Snippet: The following day, the cells were transfected with plasmids encoding GSK3β WT (Addgene, #14753), GSK3β S9A (Addgene, #14754), or GSK3β K85A (Addgene, #14755) using Lipofectamine LTX (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Western Blot

Journal: iScience

Article Title: PERIOD 2 regulates low-dose radioprotection via PER2/pGSK3β/β-catenin/Per2 loop

doi: 10.1016/j.isci.2022.105546

Figure Lengend Snippet: GSK3β/β-catenin pathway is involved in Per2 mediated adaptive radioprotection (see also Figure S6 ) (A) Correlation of Per2 and GSK3β in human mammalian tissue analyzed with the GEPIA database. Pearson correlation analysis, R = Pearson correlation coefficient. (B) Correlation of Per2 and β-catenin in human mammalian tissue analyzed with the GEPIA database. Pearson correlation analysis, R = Pearson correlation coefficient. (C) Left, western blot of phosphorylated GSK3β (Ser9), AKT, p -AKT and active β-catenin in Per2 wt and Per2 def BMMNCs; right, relative expression of indicated proteins quantified with ImageJ and normalized with β-actin levels. Data are represented as mean ± SEM, n = 3, ∗∗p < 0.01, ns p > 0.05, Student’s t test. (D) Western blot of phosphorylated GSK3β (Ser9) in Per2 wt /GSKß wt MEFs following exposure to LDR with estimated GSK3β phosphorylation peak at 12 h. (E) Cell apoptosis of Per2 wt /GSKß wt MEFs compared to Per2 wt /GSKß ko MEFs after treated with LDR (10 cGy), HDR (5 Gy), or LDR 12 h before HDR. Data are represented as mean ± SEM, n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001, ns p > 0.05, ANOVA two-way test was applied. (F) Western blot of pGSK3β(Ser9), GSK3β, and active β-catenin in Per2 wt and Per2 def mice BMMNCs 12 h after sham and LDR. (G) Relative expression of pGSK3β and active β-catenin in Per2 wt and Per2 def mice BMMNCs 12 h after LDR was quantified with ImageJ and normalized with β-actin levels. Data are represented as mean ± SEM, n = 3, ∗∗∗p < 0.001, ns p > 0.05, Student’s t test. (H) Western blot of pGSK3β(Ser9), GSK3β, active β-catenin, and Per2 in Per2 wt /GSK3ß wt and Per2 wt /GSK3ß ko MEFs 12 h after sham and LDR. (I) Relative expression of PER2 and active β-catenin in Per2 wt /GSK3ß wt and Per2 wt /GSK3ß ko MEFs 12 h after sham and LDR was quantified with ImageJ and normalized with β-actin levels. Data are represented as mean ± SEM, n = 3, ∗p < 0.05, ∗∗p < 0.01, ns p > 0.05, ANOVA two-way test was applied.

Article Snippet: GSK3β wt and GSK3β S9A-mut plasmids were derived from Addgene and transfected with the Per2-OE 293T cells following the protocol of turbo reagent transfection (Thermo Scientific, Cat. R0531).

Techniques: Western Blot, Expressing, Phospho-proteomics

Journal: iScience

Article Title: PERIOD 2 regulates low-dose radioprotection via PER2/pGSK3β/β-catenin/Per2 loop

doi: 10.1016/j.isci.2022.105546

Figure Lengend Snippet:

Article Snippet: GSK3β wt and GSK3β S9A-mut plasmids were derived from Addgene and transfected with the Per2-OE 293T cells following the protocol of turbo reagent transfection (Thermo Scientific, Cat. R0531).

Techniques: Western Blot, Immunoprecipitation, Recombinant, Luciferase, Transfection, Library Quantification, ATP Assay, Bicinchoninic Acid Protein Assay, Software, Control, Sequencing, Plasmid Preparation, Gene Expression, Microscopy