Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: 12B2 and 15C2 are specific for nonphospho-Ser GSK3β/α peptides. Each antibody was screened in indirect ELISA titers against npS9 GSK3β, pS9 GSK3β, npS21 GSK3α and pS21 GSK3α peptides ( n = 3 independent experiments). (A) 12B2 showed strong reactivity for npS9 GSK3β compared to npS21 GSK3α peptides and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.1 nM; pS9 = indeterminate (id); npS21 = 6.4 nM; pS21 = id). (B) To further confirm the specificity of 12B2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 12B2 showed strong reactivity with the npS GSK3 peptides (β > α), but did not react with pS GSK3 peptides. (C) 15C2 showed stronger reactivity for npS21 GSK3α compared to npS9 GSK3β and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.4 nM; pS9 = id; npS21 = 277 pM; pS21 = id). (D) To further confirm the specificity of 15C2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 15C2 showed strong reactivity with the npS GSK3 peptides, but did not react with pS GSK peptides. It is noteworthy that synthetic peptides provide a homogeneous source of modified peptides, and thus, are ideal for challenging the specificity of the antibodies against nonphospho-Ser and phospho-Ser residues in GSK3.

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Indirect ELISA, Modification

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: 12B2 is specific for nonphospho-S9 recombinant GSK3β, and 15C2 is specific for nonphospho-S9/21 recombinant GSK3β/α. (A–C) Western blots of recombinant GSK3β and α alone (input, In), phosphorylated S9 GSK3β and S21 GSK3α proteins (Ph; i.e., Akt1 treated) and dephosphorylated S9 GSK3β and S21 GSK3α proteins (NP; i.e., alkaline phosphatase treated). The GSK3α is GST-tagged and GSK3β is his-tagged, and in each blot the 12B2 (A) , 15C2 (B) or pS9 GSK3β (C) antibodies are red, while total GSK3α/β antibody is green. (D) Quantitation of the 12B2 blots shows strong reactivity with npS9 GSK3β, and none with npS21 GSK3α, pS9 GSK3β or pS21 GSK3α. (E) Quantitation of the 15C2 blots shows strong reactivity with npS9 GSK3β and npS21 GSK3α, but none with pS9 GSK3β or pS21 GSK3α. (F) Quantitation of the pS9 GSK3 blots confirm that the Akt1 treatment produced robust phosphorylation of S9 in GSK3β (and S21 in α) and that the alkaline phosphatase treatment removed phosphorylation of S9 in GSK3β (and S21 in α). Each experiment was repeated three-four independent times and all samples were loaded at 50 ng GSK3/lane. The data are normalized to total GSK3 signal.

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Recombinant, Western Blot, Quantitation Assay, Produced

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: 12B2 and 15C2 are specific for nonphospho-S GSK3 in brain lysates of human, mouse, and rat, and both effectively immunoprecipitate GSK3 from cell lysates. (A) Protein sequence alignments for GSK3β (amino acids 1–25) and GSK3α (amino acids 13–37) from human, mouse and rat (Uniprot IDs in parentheses). (B,C) Blots of lysates from human, mouse and rat cortical tissue and GAPDH was used as a loading control (40 μg/lane total protein loaded; experiment repeated three times). (B) 12B2 (red) specifically labeled GSK3β, not GSK3α, in lysates and total GSK3α/β (green) was used to identify both isoforms. (C) 15C2 (red) labeled both GSK3β and GSK3α in lysates and total GSK3α/β (green) was used to identify both isoforms. (D–F) The 12B2 (D) , 15C2 (E) , or control mouse IgG ( F , Ms IgG) were used to immunoprecipitate GSK3 enzymes from HEK293T cell lysates. The starting lysate (Input) was incubated with magnetic beads coated with 12B2 (D) , 15C2 (E) , or Ms IgG control (F) antibodies. 12B2 pulled down only GSK3β (12B2-IP), 15C2 pulled down both GSK3α and β (15C2-IP) and Ms IgG did not pull down GSK3α or β (MsIgG-IP). The post-IP lysates were also run for comparisons to the input samples. These experiments were performed three independent times.

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Sequencing, Labeling, Incubation, Magnetic Beads

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: GSK3β antibody immunostaining in cultured cells and tissue sections. (A,B) HEK293T cells stained with 12B2 ( A , green) and 15C2 ( B , green). ( C,D ) Undifferentiated SH-SY5Y cells stained with 12B2 ( C , green) and 15C2 ( D , green). (E,F) Rat primary cortical neurons (E18) stained with 12B2 ( E , green) and 15C2 ( F , green). In A-F , all cells were also stained with total GSK3α/β (red) and DAPI (blue in merged image). The pattern of staining with 12B2 and 15C2 was punctate staining throughout the cells, and 12B2 produced stronger signal than 15C2 in each cell type. Scale bars = 25 μm. (G,H) Brain sections in rat (left, retrosplenial cortex displayed) and human (right, temporal cortex) stained with 12B2 (G) or 15C2 (H) . In general, both antibodies produced clear somatodendritic and parenchymal staining in human and rat brain sections. Scale bars = 50 μm.

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Immunostaining, Cell Culture, Staining, Produced

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 12B2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 12B2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 12B2 signal shows that GSK3β siRNA caused a reduction of 50% for GSK3β when compared to control cells, while GSK3α siRNA caused an increase in GSK3β (+35%). (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% for GSK3α and an increase in GSK3β (+29%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 41% for GSK3β and an increase in the GSK3α (+17%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 12B2 detection of npS9 GSK3β, which produces a punctate staining pattern, in GSK3β siRNA treated cells compared to control and GSK3α siRNA treated cells. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure .

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Quantitation Assay, Western Blot, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 15C2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 15C2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 15C2 signal shows that GSK3α siRNA caused a loss of 84% for GSK3α and an increase in GSK3β (+22%) when compared to control cells. Quantitation of 15C2 shows that GSK3β siRNA caused a loss of 49% for GSK3β and an increase in GSK3α (+18%) when compared to control. (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% in GSK3α and an increase in GSK3β (+24%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 40% for the GSK3β and an increase in GSK3α (+9%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 15C2 detection of npS21 GSK3α or npS9 GSK3β when treated with GSK3α siRNA or GSK3β siRNA, respectively. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure .

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Quantitation Assay, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: Detection of recombinant npS9 GSK3β with 12B2 and 15C2 antibodies is linear and correlates with kinase activity. (A) The level of GSK3β kinase activity with 30, 60, 120, 180, 240, and 300 ng of npS9 GSK3β (“active”) was measured using an in vitro GSK3β kinase activity assay and there was a linear increase in kinase activity with increasing amounts of GSK3β ( r 2 = 0.93). Three independent experiments were performed. (B) For western blotting, recombinant GSK3β was incubated with alkaline phosphatase to generate nonphosphoS9 GSK3β or incubated with Akt1 to generate phosphoS9 GSK3β, and then 0, 30, 60, 120, 180, 240, or 300 ng of npS9 GSK3β was mixed with 300, 240, 180, 120, 60, or 0 ng of pS9 GSK3β to bring the total protein content to 300 ng/lane. The blot was probed with 12B2 (red) and total GSK3α/β antibodies (green). (C) Quantitation of signal from 12B2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.92). (D) The same samples were probed with 15C2 (red) and total GSK3α/β antibodies (green). (E) Quantitation of signal from 15C2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.90). It is notable that both 12B2 and 15C2 signals also showed a direct correlation with GSK3β activity levels (12B2: r = 0.99, p = 0.0002; 15C2: r = 0.99, p < 0.0001). Four independent experiments were performed.

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Recombinant, Activity Assay, In Vitro, Kinase Assay, Western Blot, Incubation, Quantitation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: Treating cells with protein phosphatase inhibitor decreases npS9 GSK3β in cells. (A) A standard curve of dephosphorylated GSK3β protein captured with 12B2 antibody was used for quantitative sandwich ELISAs ( r 2 = 0.999). (B) Untreated HEK293T lysates assayed in 12B2 sandwich ELISAs at 120, 60, 30, 15, and 7.5 μg total protein/well produces a linear dose response curve ( r 2 = 0.988). Interpolation using the standard curve in (A) indicates that the lysate samples contain 7.4, 5.2, 3.5, 2.0, and 0.9 ng of npS9 GSK3β, respectively. (C) HEK293T cells were either untreated (-) or treated with 10 nM calyculin A for 30 min (+) to reduce npS9 GSK3β levels ( n = 4 independent experiments). The lysates were used in 12B2 sandwich ELISAs. A significant reduction in npS9 GSK3β levels was detected in calyculin A (10 nM) treated cells compared to untreated cells ( ∗ p < 0.05, unpaired t -test, two-tailed). The amount of npS9 GSK3β was quantitatively determined by interpolation using the recombinant GSK3β standard curve in (A) . (D) Immunofluorescence for 12B2 (green) showed an apparent qualitative reduction in npS9 GSK3β levels in HEK cells treated with calyculin A when compared to control cells. Cells were also stained with total GSK3α/β (red) and DAPI. Scale bar = 25 μm.

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Two Tailed Test, Recombinant, Immunofluorescence, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: Protein phosphatase inhibition significantly reduces GSK3β kinase activity in cells. (A) A standard curve of active GSK3β enzyme (300 – 9.4 ng) confirmed the signal in the experimental samples was within the linear range of detection in this assay ( r 2 = 0.97). Experiment was repeated three times. (B) Calyculin A treated cells showed a significant reduction in GSK3β kinase activity compared to control cells (the –TCS sample sets; all samples were used at 60 μg total protein/well). Interpolation from the recombinant GSK3β enzyme activity curve with known amounts of active GSK3β indicated that the control samples contained 29 ng of active GSK3β and calyculin A treated cells contained 15 ng. Addition of TCS-2002 (0.1 mM; +TCS), a potent GSK3β inhibitor, completely blocked kinase activity in control and calyculin A treated cells ( ∗ p < 0.05, two-way ANOVA with Holm-Sidak post hoc test, two-tailed). Note that the same lysate samples used here were used in Figure . This experiment was repeated four times.

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Inhibition, Activity Assay, Recombinant, Two Tailed Test

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: The Akt-protein phosphatase signaling pathway involved in regulating GSK3β phosphorylation. Active Akt (i.e., phosphorylated) inactivates GSK3β by phosphorylation at S9. Protein phosphatases can modulate GSK3β phosphorylation at S9 via two routes. (1) Protein phosphatases inactivate Akt by dephosphorylation, and (2) protein phosphatases activate GSK3β by directly dephosphorylating S9. Inhibition of Akt (with inhibitors such as AZD-5363) increases non-phosphorylated GSK3β by suppressing Akt-mediated phosphorylation of GSK3β. Inhibition of protein phosphatases (with inhibitors such as calyculin A) causes a decrease in non-phosphorylated GSK3β through the Akt pathway by increasing active Akt (the grayed portion of the Akt cycle). Protein phosphatase inhibition also leads to decreased non-phosphorylated GSK3β independent of Akt by directly dephosphorylating S9 in GSK3β. If an Akt inhibitor is applied followed by a protein phosphatase inhibitor the Akt-independent pathway can be evaluated.

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: De-Phosphorylation Assay, Inhibition

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: Protein phosphatases regulate GSK3β phosphorylation independent of Akt signaling. HEK293T cells were treated with an Akt inhibitor (AZD-5363, 1 μM), a protein phosphatase inhibitor (calyculin A, 10 nM) or the Akt inhibitor followed by the phosphatase inhibitor. Four independent experiments were run. (A) Western blots of samples were probed with 12B2 (npS9-GSK3β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (B) Quantitation of the blots shows that inhibition of Akt (AZD) significantly increased npS9 GSK3β, while inhibition of protein phosphatases (Caly) significantly reduced npS9 GSK3β. When Akt signaling was blocked first and then the phosphatase inhibitor was applied (AZD + Caly) a significant reduction in the level of npS9 GSK3β occurred when compared to Akt inhibitor alone. (C) Quantitation of the pS9 GSK3β blots shows an opposite pattern where inhibition of Akt significantly decreased pS9 GSK3β, while inhibition of phosphatases significantly increased pS9 GSK3β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant increase in the level of pS9 GSK3β occurred when compared to Akt treatment alone. (D) Western blots of samples were probed with 15C2 (npS9-GSK3α/β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (E,F) Quantitation of the blots shows that inhibition of Akt significantly increased npS9 GSK3α and β, while inhibition of protein phosphatases significantly reduced npS9 GSK3α and β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant reduction in the level of npS9 GSK3β and npS21 GSK3α occurred when compared to Akt inhibitor alone. Collectively, these results suggest protein phosphatases dephosphorylate Ser9/21 independent of Akt signaling. All bands are normalized to GAPDH. All groups were statistically significant from the others in ( B,C,E,F) , but only ∗ p < 0.05 vs. control and ∗∗ p < 0.05 vs. AZD alone are indicated to simplify the graph and highlight the critical comparisons (one-way ANOVA, Holm-Sidak post hoc test). Total GSK3α/β levels, total Akt and phospho-Akt immunoblots from these experiment are displayed in Supplementary Figure .

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Western Blot, Quantitation Assay, Inhibition

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

doi: 10.3389/fnmol.2016.00123

Figure Lengend Snippet: Summary of GSK3β antibody performance in assays tested in the current work.

Article Snippet: Briefly, 2 μg of recombinant GSK3β (G09-10G, Signal Chem) was incubated with 60 U alkaline phosphatase (EF0651, Fermentas) at 30° C for 2 h to create the npS9 GSK3β protein standard.

Techniques: Indirect ELISA, Activity Assay, Kinase Assay

Journal: Cell Death Discovery

Article Title: Coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and epithelial-mesenchymal transition via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3β signals

doi: 10.1038/s41420-022-01291-z

Figure Lengend Snippet: A , B The activation level of AKT/GSK3β pathway was detected by western blot in vitro and in vivo. C , D The expression levels of EMT marker molecules (N-cadherin, E-cadherin and TGFβ1), inflammatory pathway protein molecules (NLRP3, Cleaved Caspase1/Caspase1) and fibrosis marker molecules (COL-1, COL-3 and α-SMA) was detected by western blot. E Inflammatory factor IL-1β and inflammatory chemokine CXCL2 the supernatant of alveolar epithelial cells were detected by ELISA. Data were expressed as the mean ± SD, n = 3. ** P < 0.01 and *** P < 0.001. F The migration and invasion abilities of alveolar epithelial cells were detected by scratch healing assay and transwell assay.

Article Snippet: The information of antibodies is as follows: IGF1 (1: 1000, Abclonal, #A11985, China), IGF1R (1: 1000, CST, #9750, USA), p-IGF1R(Tyr1135/1136) (1: 1000, CST, #3024, USA), COL-1 (1: 2000, Abcam, ab260043, UK), COL-3 (1: 1000, Abcam, ab184993, UK), α-SMA (1:1000, CST, #19425, USA), heme oxygenase-1 (HO-1, 1: 1000, CST, #5853, USA), N-cadherin (1: 1000, CST, #49398, USA), E-cadherin (1: 1000, CST, #49398, USA), TGFβ1 (1: 1000, abclonal, #A2124, China), p-NF-kB p65(Ser536) (1: 1000, CST, #3033, USA), NF-kB p65 (1: 1000, CST, #8242, USA), NLRP3 (1: 1000, CST, #15101, USA), Cleaved Caspase1(Asp297) (1: 1000, CST, #4199, USA), Caspase1 (1: 1000, CST, #2225, USA), p-Akt(Ser473) (1: 1000, CST, #4060, USA), Akt (1: 1000, CST, #4691, USA), p-GSK3β(Ser9) (1: 1000, CST, #9322, USA), GSK3β (1: 1000, CST, #9369, USA) and β-actin (1: 1000, CST, #4970, USA).

Techniques: Activation Assay, Western Blot, In Vitro, In Vivo, Expressing, Marker, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay