Journal: Cancers

Article Title: The Tumor Suppressor Protein TRAF3 Modulates GSK3 Activity and Susceptibility of B Lymphoma Cells to GSK3 Inhibition.

doi: 10.3390/cancers14205029

Figure Lengend Snippet: Figure 1. Relationship between TRAF3 status and inactive GSK3β in B cell lymphoma cell lines. (A) Cells from BCL cell lines were lysed and GSK3β or TRAF3 was immunoprecipitated. IP samples and input/whole cell lysate (WCL) were analyzed by Western blotting. (B) Representative blot of TRAF3, GSK3β and pGSK3βS9 in a panel of BCL cell lines. (C) Graph of amount of TRAF3 vs. ratio of inactive (pGSK3β):total GSK3β from data in (B). (D) Western blot of human B cells from the peripheral blood of healthy donors. (E) Graph of amount of TRAF3 vs. ratio of inactive (pGSK3β):total GSK3β from data in (F). Representative Western blot of pGSK3βS9 and total GSK3β in primary mouse B cells from mice with or without a B cell-specific TRAF3 deficiency. (G) Graph of amount of TRAF3 vs. ratio of inactive (pGSK3β):total GSK3β from data in (F). (H) Quantification of blots in (D). Blots are representative of at least 3 independent experiments (A,B). In (C), each lane represents an individual human donor. In (D), each lane represents an individual mouse. n.s., not significant by student’s t-test. Uncropped blots and densitometry shown in Figures S3 and S4.

Article Snippet: Antibody (Ab) recognizing one of the following was added: TRAF3 (Santa Cruz Biotechnology (Dallas, TX, USA) #sc-1828, 1:1000, or Cell Signaling Technologies (CST, Danvers, MA, USA) Ab #4729, 1:100) or GSK3β (CST, #12456).

Techniques: Immunoprecipitation, Western Blot

Journal: Cancers

Article Title: The Tumor Suppressor Protein TRAF3 Modulates GSK3 Activity and Susceptibility of B Lymphoma Cells to GSK3 Inhibition.

doi: 10.3390/cancers14205029

Figure Lengend Snippet: Figure 4. Differential effect of GSK3 inhibition on IL-6-induced STAT3 activation. (A) Representative Western blot of IL-6-induced pSTAT3Y705 in 9-ING-41 or DMSO-treated mouse BCL cells (A20.2J and Traf3−/−A20.2J). (B) Quantification of 4 replicates including A. (C) Representative Western blot of IL-6-induced pSTAT3Y705 in 9-ING-41 or DMSO-treated primary WT or Traf3−/−mouse B cells. (D) Quantification of 3 replicates including C. Data points are mean ±SEM. *** p < 0.001, * p < 0.05 by repeated-measures ANOVA with Sidak’s multiple comparisons test. Uncropped blots and densitometry shown in Figures S6 and S7.

Article Snippet: Antibody (Ab) recognizing one of the following was added: TRAF3 (Santa Cruz Biotechnology (Dallas, TX, USA) #sc-1828, 1:1000, or Cell Signaling Technologies (CST, Danvers, MA, USA) Ab #4729, 1:100) or GSK3β (CST, #12456).

Techniques: Inhibition, Activation Assay, Western Blot

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Regulatory T cells ameliorate intracerebral hemorrhage-induced inflammatory injury by modulating microglia/macrophage polarization through the IL-10/GSK3β/PTEN axis

doi: 10.1177/0271678X16648712

Figure Lengend Snippet: GSK3β/PTEN axis mediated the IL-10 induced microglia polarization. (a) A representative Western-blot shows the expression of GSK3b, P-GSK3b, PTEN, and P-PTEN in microglia 40 h after co-culture. (b) The expression of GSK3β was calculated. (c) Representative Western-blot shows the expression of P-PTEN and P-GSK3β in Lentiviral shRNA-infected microglia. (d) The expression of P-PTEN was calculated (##p < 0.01 versus the vehicle; *p < 0.05, **p < 0.01 versus the microglia + Hb group; statistics were from four separate experiments with four wells per trail).

Article Snippet: Lentiviral ShRNA infection Lentiviral shRNAs directed at mouse GSK3β (sc-35525-V) and, mouse PTEN (sc-36326-V), GFP control lentiviral particles (sc-108084) and a lentivirus containing a control, non-targeting shRNA sequence (sc-108080), each with a titer of ∼5 × 10 3 infectious units/μL, were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Co-Culture Assay, shRNA, Infection

Journal: Life Science Alliance

Article Title: Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth

doi: 10.26508/lsa.201800190

Figure Lengend Snippet: (A) Western blots and quantification for total and phosphorylated GSK3β Tyr-216 Smo KO or Smo KO fibroblasts. Graph represents quantitation of three individual Western blots relative to Smo WT . N = 3, bars represent means ± SD. (B) Western blots of total and phosphorylated PTEN Thr-366 upon treatment with vehicle or MG132 in Smo KO fibroblasts. N = 3. (C, D) Western blots and quantification for total and phosphorylated PTEN Thr-366 and phosphorylated glycogen synthase (GS) Ser-614 in Smo KO or Smo KO fibroblasts at the indicated time points. Graph represents quantitation of three individual Western blots relative to Smo WT . N = 3, dots represent means ± SD.

Article Snippet: Three GSK3β inhibitors were used: SB-216763 (#sc-200646; Santa Cruz), CT99021 (#CHIR99021; Sigma-Aldrich), and AR-A014418 (#ALX-270-468-M001; Enzo Lifesciences).

Techniques: Western Blot, Quantitation Assay

Journal: Life Science Alliance

Article Title: Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth

doi: 10.26508/lsa.201800190

Figure Lengend Snippet: Genetic deletion of Smo or pharmacologic inhibition of SMO in pancreatic CAFs activates GSK3β, leading to enhanced PTEN phosphorylation and proteasomal degradation via the E3 ubiquitin ligase enzyme RNF5. Subsequent AKT activation leads to enhanced GLI2 binding and activation of the Tgfa promoter. TGF-α production by SMO-null fibroblasts cross-talks with the adjacent tumor cells, leading to activation of epidermal growth factor receptor (EGFR) and accelerated growth of the tumor epithelium.

Article Snippet: Three GSK3β inhibitors were used: SB-216763 (#sc-200646; Santa Cruz), CT99021 (#CHIR99021; Sigma-Aldrich), and AR-A014418 (#ALX-270-468-M001; Enzo Lifesciences).

Techniques: Inhibition, Phospho-proteomics, Ubiquitin Proteomics, Activation Assay, Binding Assay