Journal: bioRxiv

Article Title: Phosphoproteomics of cellular mechanosensing reveals NFATC4 as a regulator of myofibroblast activity

doi: 10.1101/2023.02.13.528335

Figure Lengend Snippet: Pathway and upstream regulator analysis reveals stiffness regulated processes. A. Heat map shows enriched terms across gene lists of clusters 1–10 in main , colored by p-values. Enrichment analysis was performed with Metascape ( www.metascape.org ). B. The graphs show CK2A1 targets predicted by Ingenuity Pathway Analysis (IPA) and their phosphorylation status in the phosphoproteomic data set. Turquoise color shades indicate low phosphorylation and pink color shades indicate high phosphorylation. Doted lines indicate an indirect interaction and continuous lines specify a direct interaction from prior knowledge databases. C. Results of pathway enrichment analysis of the IPA CK2A1 target sites using Fisher exact test (FDR < 5%). D. Immunoblot probed with an antibody specific for proteins containing a pS/pTDXE motif, which represents a CK2 phosphorylation consensus sequence. GAPDH served as a loading control (n=3).

Article Snippet: Antibodies used were: Anti-GAPDH (1:1000, Cell Signaling #5174S), Anti-CK II alpha (1:1000, Abcam ab70774), Anti-Phospho-CK2 Substrate Motif [(pS/pT)DXE] (1:1000, Cell Signaling #8738), Anti-Phospho-Myosin Light Chain 2 (Thr18/Ser19) (1:2000, Cell Signaling #3674), Anti-Phospho-FoxO3a (S7) (1:1000, Cell Signaling 14724S), Anti-Phospho-Paxillin (S126) (1:1000, Life Technologies 441022G), Anti-Phospho-CBX3 (Ser93) (1:1000, Cell Signaling #2600), Anti-rabbit IgG, HRP-linked (1:20,000, Cell Signaling #7074) Anti-mouse IgG, HRP-linked (1:20,000, Cell Signaling #7074).

Techniques: Western Blot, Sequencing

Journal: PLoS ONE

Article Title: Stearoyl-CoA Desaturase-1 (SCD1) Augments Saturated Fatty Acid-Induced Lipid Accumulation and Inhibits Apoptosis in Cardiac Myocytes

doi: 10.1371/journal.pone.0033283

Figure Lengend Snippet: (A): FA oxidation was significantly increased in rat neonatal cardiac myocytes treated with 100 µM palmitic acid (PA). (B): On the other hand, glucose oxidation was significantly reduced in cardiac myocytes treated with PA (100 µM). (C): PA (50 to 100 µM) induced excessive FA oxidation was attenuated in cardiac myocytes transduced with a MOI of 20 of Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). (D): PA (50 to 100 µM) -induced suppression of glucose oxidation recovered in cardiac myocytes transduced with Ad-SCD1 (▪) in comparison with control cells transduced with Ad-LacZ (□). BSA was used as a vehicle. Each sample was counted in a scintillation counter (cpm) and data are shown as the mean ± SD. **P<0.01 vs. Ad-LacZ in each PA concentration. (E); Phosphorylation levels of AMPK and ACC decreased in cardiac myocytes transduced with an MOI of 20 of Ad-SCD1 in comparison with control cells transduced with Ad-LacZ. Oligomycin (1 µM) was used as an AMPK and ACC activator.

Article Snippet: The mixture was rotated at 4°C for 15 min and centrifuged at 14000 rpm for 15 min. Western blot analysis was performed according to standard procedures using the following primary antibodies: rabbit monoclonal phosphor-AMPK, AMPK, phosphor-ACC, ACC, cleaved-caspase 3, caspase 3 mAb (Cell Signaling) and β-actin (Santa cruz biotechnology, CA, USA).

Techniques: Transduction, Concentration Assay

Journal: PLoS ONE

Article Title: Geminin Is Required for Zygotic Gene Expression at the Xenopus Mid-Blastula Transition

doi: 10.1371/journal.pone.0038009

Figure Lengend Snippet: (A) The signaling pathway that controls entry into mitosis. See text for details. (B) Arrested Geminin-deficient cells exhibit increased phosphorylation of Cdc2 on Y 15 and increased levels of B-type cyclins; and these changes are reversed by over-expressing either Cdc2 AF or Cdc25 S287A . Two-cell embryos were left uninjected (CO), injected on both sides with anti-Gem MOs (a-Gem MOs), or injected with anti-Gem MOs followed by RNA encoding Cdc2 AF or Cdc25 S287A . At stage 10.5, phosphorylated Cdc2 and cyclin B1 levels were determined by immunoblotting. Load, cross-reacting band serving as a loading control. (C) The cell cycle arrest is reversed by over-expressing Cdc25 WT , Cdc25 S287A , Cdc2 AF , or Chk1 DA . One cell of a two-cell embryo was injected with anti-Geminin MOs and RNA encoding the indicated proteins. The plot shows the percentage of embryos in which cell division was restored in the injected area at stage 10.5. (D–M) One cell of a two-cell embryo was injected with anti-Gem MOs and/or RNA encoding the indicated proteins. LacZ RNA was co-injected as a lineage tracer. At stage 10.5 Xbra RNA was visualized by in situ hybridization (purple) and beta galactosidase activity was visualized by staining with Xgal (blue). (N) Both sides of a two-cell embryo were injected with anti-Geminin MOs and/or RNA encoding the indicated proteins. At stage 10.5, RNA was extracted and the amount of Xbra mRNA was measured by RT-PCR (gray bars). In a parallel experiment, one cell of a 2-cell embryo was injected in the same way along with LacZ as a lineage tracer. At stage 10.5 Xbra was visualized by in situ hybridization and the percentage of embryos showing normal Xbra expression was determined (black bars).

Article Snippet: Phosphospecific antibodies against pS 139 H2A.X and pS 345 Chk1 were purchased from Cell Signaling.

Techniques: Expressing, Injection, Western Blot, In Situ Hybridization, Activity Assay, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Geminin Is Required for Zygotic Gene Expression at the Xenopus Mid-Blastula Transition

doi: 10.1371/journal.pone.0038009

Figure Lengend Snippet: Two-cell Xenopus embryos were injected on both sides with anti-Geminin MOs and/or RNA encoding the indicated proteins. The levels of S 345 -phosphorylated Chk1, S 139 -phosphoryated H2A.X, and Geminin were determined by immunoblotting.

Article Snippet: Phosphospecific antibodies against pS 139 H2A.X and pS 345 Chk1 were purchased from Cell Signaling.

Techniques: Injection, Western Blot

Journal: PLoS ONE

Article Title: Geminin Is Required for Zygotic Gene Expression at the Xenopus Mid-Blastula Transition

doi: 10.1371/journal.pone.0038009

Figure Lengend Snippet: Two-cell Xenopus embryos were injected on both sides with anti-Geminin MOs and/or RNA encoding the indicated proteins. The levels of S 345 -phosphorylated Chk1, S 139 -phosphoryated H2A.X, and Geminin were determined by immunoblotting.

Article Snippet: Phosphospecific antibodies against pS 139 H2A.X and pS 345 Chk1 were purchased from Cell Signaling.

Techniques: Injection, Western Blot

Journal: PLoS ONE

Article Title: Btk Regulates Macrophage Polarization in Response to Lipopolysaccharide

doi: 10.1371/journal.pone.0085834

Figure Lengend Snippet: (A) WT and Btk −/− BMDMs were treated ex vivo with LPS (100 ng/ml) or IFN-γ (100 U/ml) for 3 hours or 15 minutes, respectively, lysates were prepared and phosphorylated Y701-STAT1 levels determined by Western blot. WT and Btk −/− BMDMs were treated ex vivo with an M1 or M2 polarizing cocktail over the indicated time course, lysates were prepared and tyrosine phosphorylated STAT1 and STAT6 (B), serine phosphorylated AKT (upper panel) and NF-κB p65 (lower panel) (C) iNOS and SHIP-1 (D) levels were determined by Western blot. Results in each case are representative of three independent experiments. Densitometric analysis was performed and graphs represent phosphorylated protein levels relative to unphosphorylated proteins (B-C) or changes in total protein levels relative to β-actin (D) for WT and Btk −/− BMDMs. Student’s paired t test was performed comparing relative expression of phosphorylated or total proteins in Btk −/− BMDMs to WT BMDMs following treatment with polarizing cocktails as indicated. Results shown are mean±SD from three independent experiments. * = p≤0.05.

Article Snippet: Expression of STAT1(Santa Cruz Biotechnology #sc-592), STAT6 (Santa Cruz Biotechnology #sc-621), pY-STAT1 (Cell Signaling #9171), pY-STAT6 (Imgenex #IMG408A), Akt (Cell Signaling #9271), pS-Akt (Cell Signaling #9272), NF-κB p65 (Santa Cruz Biotechnology #sc-372), pS-NF-κB p65 (Cell Signaling #3036S), iNOS (Transduction Laboratories #N39120) and SHIP-1 (Santa Cruz Biotechnology #sc8425), was determined by Western blot as described previously .

Techniques: Ex Vivo, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Btk Regulates Macrophage Polarization in Response to Lipopolysaccharide

doi: 10.1371/journal.pone.0085834

Figure Lengend Snippet: (A) This study has demonstrated that in response to LPS and IFN-γ Btk contributes to M1 polarizing of myeloid cells via promoting the phosphorylation of Akt and subsequently the p65 subunit of NFκB, in addition to enhancing to phosphorylation of STAT1. (B) In the absence of Btk exposure of myeloid cells to LPS and IFN-γ results in the preferential induction of M2 associated genes and preferentially recruitment of M2 cells in vivo . Previous studies in Btk −/− mice have observed increased levels IL-10 systemically following LPS treatment. IL-10 is known to activate STAT3 and there is some evidence to suggest that STAT3 may also play a role in promoting M2 macrophage polarization. Thus in the absence of Btk, in response to M1 polarizing stimuli increased IL-10 production together with reduced phosphorylation of key signaling intermediaries, combined with activation of p50 the inhibitory subunit of NF-κB could potentially account for the observed preferential skew towards an M2 phenotype. Additionally this study has shown that in response to IL-4 and IL-13 Btk −/− cells demonstrate an increased capacity to polarize towards an M2 phenotype as a result of enhanced STAT6 phosphorylation and increased SHIP1 expression.

Article Snippet: Expression of STAT1(Santa Cruz Biotechnology #sc-592), STAT6 (Santa Cruz Biotechnology #sc-621), pY-STAT1 (Cell Signaling #9171), pY-STAT6 (Imgenex #IMG408A), Akt (Cell Signaling #9271), pS-Akt (Cell Signaling #9272), NF-κB p65 (Santa Cruz Biotechnology #sc-372), pS-NF-κB p65 (Cell Signaling #3036S), iNOS (Transduction Laboratories #N39120) and SHIP-1 (Santa Cruz Biotechnology #sc8425), was determined by Western blot as described previously .

Techniques: In Vivo, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: APP Is Cleaved by Bace1 in Pre-Synaptic Vesicles and Establishes a Pre-Synaptic Interactome, via Its Intracellular Domain, with Molecular Complexes that Regulate Pre-Synaptic Vesicles Functions

doi: 10.1371/journal.pone.0108576

Figure Lengend Snippet: Analysis of γ -secretase subunits distribution in brain fractions isolated from WT mice. SV, P43 and SP fractions were prepared using the protocol described in and analyzed for the presence of the γ -secretase components Ps-1, Ps-2, Nct and Pen2 using Western blots. While all four γ-secretase components were readily detected in the P43 and, albeit at lower levels, SP fractions, only Ps-1 was detected, at very minimal level, in the SV fraction. Gapdh was used to normalize the loading.

Article Snippet: The following antibodies were used in Western blots: anti-APP C-terminal AbD (Zymed), anti-APP N-terminal 22C11 (Millipore), anti-sAPPβ (antibodies-online ABIN927102), anti-APPpThr 668 (Cell Signaling Technology), anti-Bace1 (Cell Signaling Technology), anti-Ps-1 (Cell Signaling Technology), anti-Ps-2 (Cell Signaling Technology), anti-Pen2 (Cell Signaling Technology), anti-Nicastrin (Cell Signaling Technology), anti-Synaptotagmin (Sigma-Aldrich), anti-Synaptophysin (Cell Signaling Technology), anti-Rab3A (Cell Signaling Technology), anti-Synaptobrevin/Vamp2 (Synaptic System), anti-Vdac (Cell Signaling Technology), Rab4 (Cell Signaling Technology), transferrin receptor (Sigma-Aldrich), anti-NmdaR2A (Cell Signaling Technology), anti-NmdaR2B (Cell Signaling Technology), anti-Gapdh (Cell Signaling Technology), anti-Nsf (Cell Signaling Technology), anti-Snap25 (Cell Signaling Technology), anti-Sx1b (Synaptic System).

Techniques: Isolation, Western Blot