Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of RACK1-Mediated NLRP3 Oligomerization (Active Conformation) Ameliorates Acute Respiratory Distress Syndrome.

doi: 10.1002/advs.202411355

Figure Lengend Snippet: Figure 7. Bigelovin targets the RACK1 protein. A) Workflow of isoDTB-ABPP experiment. B) Bigelovin binding proteins identified by isoDTB-ABPP experiment. C–E) LPS-primed BMDMs were incubated with DMSO or bigelovin (300 nM) for 1 h, and cellular thermal shift assays (CETSA) analyzed the thermal stabilization of Agfg2 (C), Sash1 (C), Gbp4 (D), RACK1 (E) at different temperatures (n = 3). F) LPS-primed BMDMs were incubated with different concentration of bigelovin for 1 h, then thermal stability of RACK1 protein was evaluated by CESTA at 49 °C (n = 3). G) Mass spectrometry analysis of the binding site of bigelovin to RACK1. H) HEK-293T cells transfected with indicated plasmid were incubated with bigelovin (300 nM) for 24 h, then thermal stability of GFP-RACK1 and GFP-RACK1 (C168A) protein were evaluated by CESTA at different temperature (n = 3). I) Mice were administrated with DMSO or bigelovin (0.1 mg kg−1) for 3 days by intraperitoneal injection and then challenged with 1% starch broth solution for 48 h, thermal stability of RACK1 protein in peritoneal macrophages was evaluated by CESTA at different temperature (n = 3). J) The interaction of bigelovin with RACK1 was measured by SPR. K) The interaction between bigelovin and RACK1 was measured by molecular docking.

Article Snippet: Surface Plasmon Resonance: The binding affinity between bigelovin and recombinant human RACK1 protein (Biorbyt, orb754920) was assayed using a WeSPR One instrument.

Techniques: Binding Assay, Incubation, Concentration Assay, Mass Spectrometry, Transfection, Plasmid Preparation, Injection, Starch

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of RACK1-Mediated NLRP3 Oligomerization (Active Conformation) Ameliorates Acute Respiratory Distress Syndrome.

doi: 10.1002/advs.202411355

Figure Lengend Snippet: Figure 8. Bigelovin inhibits NLRP3 inflammasome by binding to cysteine 168 of RACK1. A,B) LPS-primed BMDM was treated with bigelovin and then stimulated with ATP (5 mM, 30 min). Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies (A). Quantitative analysis of the interaction of NLRP3-RACK1 described in (A) (n = 3) (B). C,D) HEK-293T cells were transfected FLAG-NLRP3 with GFP-Vector or GFP-RACK1 and treated with bigelovin (300 nM) for 24 h. Cell lysates were immunoprecipitated and immunoblotted for analysis of the interaction of FLAG-NLRP3 and GFP-RACK1 (C). Quantitative analysis of the interaction of GFP-RACK1-FLAG-NLRP3 described in (C) (n = 3) (D). E,F) HEK-293T cells were transiently transfected with and either GFP-RACK1 or GFP-RACK1 (C168A) and treated with bigelovin (300 nM) for 24 h. Samples were immunoprecipitated with FLAG and probed for FLAG (NLRP3) and GFP (RACK1 or RACK1 (C168A) (E). Quantitative analysis of the immunoblot of GFP-RACK1/FLAG-NLRP3 described in 9(E) (n = 3) (F). G,H) Immunoprecipitation analysis of NLRP3-NLRP3 interaction in HEK-293T cells transfected with FLAG-NLRP3, GFP- NLRP3, GFP-RACK1 or GFP-RACK1 mutant (C168A) plasmid and treated with bigelovin (300 nM) for 24 h (G). Quantitative analysis of the interaction GFP-NLRP3-FLAG-NLRP3 described in (G) (n = 3) (H). I,J) Mice were intravenously injected with siRNA (80 μg per mouse) with in vivo-jetPEI, followed by intraperitoneal injection of 7.5 mg kg−1 LPS 48 h later, and then injected intraperitoneally bigelovin for 12 h. I) Western blotting analysis of RACK1 and caspase-1 (p12) from above mice (n = 6). J) ELISA quantification of IL-1𝛽levels of lung tissues from above mice were measured (n = 6). Data were presented as mean ± SEM and statistical significance was assessed by two-tailed unpaired t test.

Article Snippet: Surface Plasmon Resonance: The binding affinity between bigelovin and recombinant human RACK1 protein (Biorbyt, orb754920) was assayed using a WeSPR One instrument.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Injection, In Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell reports

Article Title: LYSMD3: A mammalian pattern recognition receptor for chitin

doi: 10.1016/j.celrep.2021.109392

Figure Lengend Snippet: (A) Direct binding of recombinant (r) LYSMD3 to immobilized carbohydrates or bovine serum albumin (BSA), determined by ELISA. The plate was coated with chitin DP5, DP7, LPS-EB, or BSA. An increasing amount ectodomain of LYSMD3 was added, and the amount bound was detected with the LYSMD3 antibody. See also . (B) Direct binding of rLYSMD3 to chitin beads. A mixture of purified ectodomain of LYSMD3 and BSA was incubated with chitin magnetic beads. LYSMD3 bound chitin, while BSA remained in the soluble fraction (Unbound). Samples were assayed by SDS-PAGE and Coomassie blue stain. (C) rLYSMD3 (150 ng) was incubated with chitin beads in the presence of chitin DP6 (3 mM). Bound LYSMD3 was detected by western using anti-LYSMD3. rLYSMD3 was run in the first two lanes. Western bands were quantified by densitometry using VisionWorks 9. Binding was normalized to the value without carbohydrate as 100%. (D) rLYSMD3 and GFP were incubated (2.5 μg per sample) with suspensions of 1 × 10 7 heat-killed C. albicans SC5314 (HK Cα ). Proteins bound to fungal cell wall were detected by immunoblotting with anti-LYSMD3 or anti-GFP antibody. rLYSMD3 and GFP were run in the first two lanes. (E) rLYSMD3, TLR2, TLR4 and GFP were incubated (2.5 μg per sample) with suspensions of 5 × 10 6 live Alternaria alternata (Alt) spores. Proteins bound to fungal cell wall were detected by immunoblot with anti-LYSMD3, anti-TLR2, anti-TLR4, or anti-His tag (GFP) antibody. rLYSMD3, TLR2, TLR4, and GFP were run in the first two lanes. (F) Binding of rLYSMD3 to immobilized β-glucans or BSA by ELISA. See also - . (G) 100 ng of rLYSMD3 ectodomain was incubated with heat-killed C. albicans SC5314 (HK Cα ) or A. alternata spores in the presence of soluble WGP, laminarin, or chitin DP6 (2 mg/mL). Bound LYSMD3 was detected by western and quantified by densitometry. Binding was normalized to the value without carbohydrate as 100%. Data are from one representative of three independent experiments (mean and SD of three samples per group in A and F).

Article Snippet: Alternaria alternata (ATCC 66981) was cultured on potato dextrose agar (0.4% potato starch, 2% dextrose, 1.5% agar) and incubated at 25°C in the absence of light.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Incubation, Magnetic Beads, SDS Page, Staining, Western Blot

Journal: Cell reports

Article Title: LYSMD3: A mammalian pattern recognition receptor for chitin

doi: 10.1016/j.celrep.2021.109392

Figure Lengend Snippet: (A) ELISA of cytokines in supernatants of BEAS-2B cells stimulated for 24 h with 500 μM chitin oligomers. (B) Immunoblot of LYSMD3 in membrane protein fraction of BEAS-2B cells 48 h after transfection with control siRNA or a siRNA pool specific for LYSMD3. NaK ATPase is loading control. See also . (C) ELISA of cytokines in supernatants of BEAS-2B cells transfected with siRNAs as in (B), then stimulated for 24 h with chitin DP7 (500 μg/mL), chitin particles (500 μg/mL), Alternaria spores (2 × 10 5 /mL), FLA-ST (300 ng/mL), or Poly(I:C) (3 μg/mL). See also and . (D) Immunoblot of LYSMD3 in cell lysates of NHBE cells 48 h after transfection with control siRNA or siRNA specific for LYSMD3. β-Actin is loading control. (E) ELISA of IL-8 in supernatants of NHBE cells transfected with siRNA as in (D) and then stimulated for 24 h with chitin DP7 (500 μg/mL) or A. alternata spores (2 × 10 5 /mL). (F) Immunoblot of LYSMD3 in membrane protein fraction of RAW 264.7 cells 48 h after transfection with control siRNA or siRNA specific for LYSMD3. (G) ELISA of IL-6 in supernatants of RAW 264.7 cells transfected with siRNA as in (F) and then stimulated for 24 h with chitin DP7 (100 μg/mL) or Pam3CSK4 (100 ng/mL). (H) MTT assay 48 h after transient transfection of siRNA asin(B)in BEAS-2B cells. Cell growth rate is expressed as absorbance at 570 and was normalized to the value with control siRNA as 1. (I) LDH cytotoxicity assay 48 h after transient transfection of siRNAs as in (B) in BEAS-2B cells. (J) Expression of LYSMD3 protein in BEAS-2B cell with CRISPR-Cas9 knockout of LYSMD3 (sgD3). Hsp90 is the loading control. (K) ELISA of IL-8 in supernatants of BEAS-2B cell knockout of LYSMD3 as in (J), unstimulated or stimulated for 24 h with chitin DP7 (500 μg/mL) or FLA-ST (300 ng/mL). See also . (L) ELISA of IL-33 in supernatants of hBE33 cells transfected with siRNA as in (D) and then stimulated for 1 h with Alternaria extract (200 μg/mL). *p < 0.05, **p < 0.01, and ***p < 0.001 (Student’s t test). Data are representative of three experiments (mean and SD of three samples per group in A, C, and E, or four samples per group in G, K, and I, or eight samples per group in H and L).

Article Snippet: Alternaria alternata (ATCC 66981) was cultured on potato dextrose agar (0.4% potato starch, 2% dextrose, 1.5% agar) and incubated at 25°C in the absence of light.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Membrane, Transfection, Control, MTT Assay, LDH Cytotoxicity Assay, Expressing, CRISPR, Knock-Out

Journal: Cell reports

Article Title: LYSMD3: A mammalian pattern recognition receptor for chitin

doi: 10.1016/j.celrep.2021.109392

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Alternaria alternata (ATCC 66981) was cultured on potato dextrose agar (0.4% potato starch, 2% dextrose, 1.5% agar) and incubated at 25°C in the absence of light.

Techniques: Control, Recombinant, Magnetic Beads, Transfection, Enzyme-linked Immunosorbent Assay, Membrane, Protein Extraction, Isolation, MTT Cell Proliferation, LDH Cytotoxicity Assay, Software

Journal: World Journal of Gastroenterology : WJG

Article Title: miRNA studies in in vitro and in vivo activated hepatic stellate cells

doi: 10.3748/wjg.v17.i22.

Figure Lengend Snippet: Upregulated genes in miR-146a-transfected hepatic stellate cell-2

Article Snippet: Taqman assays used were smooth muscle α-actin (SMAA) (Rn01759928_g1), Col1a1 (Rn01463849_g1), interleukin (IL)-6 (Rn00561420_m1), cyclooxygenase-2 (Cox-2) (Rn00568225_m1), RelA (Rn01502266_m1), CD31 (Rn01467259_m1), Albumin (Rn01413833_m1), CD68 (Rn01495643_g1) and tissue inhibitor of metalloproteinase (TIMP)-3 (Rn00441826_m1).

Techniques: Membrane, Translocation Assay, Coagulation, Sequencing, Binding Assay, Clinical Proteomics, Protein Binding, Activity Assay, RNA Binding Assay, Starch

Journal: Nature metabolism

Article Title: Spatial hepatocyte plasticity of gluconeogenesis during the metabolic transitions between fed, fasted and starvation states

doi: 10.1038/s42255-025-01269-y

Figure Lengend Snippet: ( a, b ) Compass-score differential activity analysis of the Vitamin A pathway between 0 h and 8 h pericentral/periportal hepatocytes ( A ) and between 0 h and 24 h pericentral/periportal hepatocytes ( B ) based on Cohen’s D analysis. BCDO (BCMO1) is highlighted. ( c, d ) Compass-score differential activity analysis of the Vitamin B6 pathway between 0 h and 8 h pericentral/periportal hepatocytes ( C ) and between 0 h and 24 h pericentral/periportal hepatocytes ( D ) based on Cohen’s D analysis. PNPO is highlighted. ( e, f ) Compass-score differential activity analysis of the Starch and sucrose (glycogen) pathway between 0 h and 8 h pericentral/periportal hepatocytes ( E ) and between 0 h and 24 h pericentral/periportal hepatocytes ( F ) based on Cohen’s D analysis. GLPASE (PYGL) is highlighted. ( g ) Representative immunoblotting of BCMO1, PNPO, PYGL, CYP2E1, E-Cadherin, and Vinculin from pericentral and periportal isolated hepatocytes.

Article Snippet: Commercially available primary antibodies were purchased for the detection of PEPCK (Abcam, no. ab70358; 1:2,000), CYP2E1 (Invitrogen, no. PA5–52652; 1:1,000), glutamine synthetase (Invitrogen, no. MA5–27749; 1:8,000), E-cadherin (Cell Signaling Technology, no. 3195; 1:2,000), GLS2 (Abcam, no. ab113509; 1:1,500), BCMO1 (Invitrogen, no. PA5–90759; 1:1,000), PNPO (Proteintech, no. 15552–1-AP; 1:1,000), PYGL (Santa Cruz, no. sc-517597; 1:1,000) and vinculin (Santa Cruz, no. sc-73614; 1:1,000).

Techniques: Activity Assay, Starch, Western Blot, Isolation

Journal: bioRxiv

Article Title: A sucrose non-fermenting-1-related protein kinase 1 gene from wheat, TaSnRK1α regulates starch biosynthesis by modulating AGPase activity

doi: 10.1101/2023.07.01.547320

Figure Lengend Snippet: (A) TaSnRK1 α truncated sequence lacking either ATP-binding domain or UBA domain were generated by PCR mediated deletion approach and transformed into pYES2. Spotting assay was performed on minimal media lacking uracil in presence of either glucose or sucrose. Growth of serially diluted cultures was assessed after incubating at 30 °C for 3 days. Ura/DO; Uracil dropout media (B) Sub-cellular localization of 35S:TaSnRK1α:GFP recombinant protein was assessed in tobacco leaves. Agrobacterium transformed 35S:TaSnRK1α:GFP was infiltrated into the 4-week-old tobacco leaves, and GFP signal was observed under confocal microscopy after 2 days of infiltration. An empty vector was used as a positive control. All the GFP images were taken with a gain % of 700 to maintain consistency. (C) Immunoblot showing TaSnRK1α:GFP fused protein abundance in the 35S:TaSnRK1α:GFP infiltrated leaves and control leaf with empty vector. Wheat anti-TaSnRK1α specific antibody was used for immunoblot assay in 1:5000 dilution and ponceaus S Staining shows the equal intensity Rubisco in the western blot. (D) Total starch content in transiently expressed TaSnRK1α in N. benthamiana leaves. Total starch was quantified using enzymatic digestion from two independent leaves (O/E 1 and O/E 2). An empty vector infiltrated leaf was used as an internal control. Error bars denote standard deviation. (Ordinary One-way ANOVA; ***, p < 0.001; n=3). O/E: TaSnRK1α over-expression.

Article Snippet: To analyse, the sub-cellular localization of wheat SnRK1, the full length CDS of TaSnRK1 α was cloned into the plant expression vector pCAMBIA1302 without stop codon under 35S promoter, with GFP protein fused at C- terminal and NcoI restriction enzyme sites on both ends using Gibson Assembly (NEB, USA).

Techniques: Sequencing, Binding Assay, Generated, Transformation Assay, Spotting Assay, Recombinant, Confocal Microscopy, Plasmid Preparation, Positive Control, Western Blot, Control, Staining, Starch, Standard Deviation, Over Expression

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 1. The macrophage-stimulating activity of ESG. (A and B) Participation of the macrophage in ESG activity. CD11b+ and CD11b−cells (1 × 106 per 1 mL) derived from BALB/c PEC were incubated with saline, ESG (50–500 μg/mL) or Pam3CSK4 (TLR2 ligand; 10 ng/mL) in the presence of IFN-γ (10 ng/mL) for 48 h, then NO (A) and IL-6 (B) concentrations in the supernatants were determined. (C and D) Role of IFN-γ in the macrophage activation by ESG. CD11b+

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Derivative Assay, Incubation, Saline, Activation Assay

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 3. The activity of ESG on TLR2-expressing HEK293 cells. HEK293 cells transfected with TLR2 expression plasmid and reporter plasmids for 24 h were stimulated by either Pam3CSK4 (0–5 ng/mL), ESG (0–400 μg/mL), LPS (0–1000 ng/mL) or CpG-ODN (TLR9 ligand; 0–1000 ng/mL) at each indicated concentration for 24 h. The relative activity was calculated as the ratio of pNF-κB-Luc (firefly) activity to RL-TK (Renilla) activity. Values are the mean ± SD. Data are representative of three independent experiments (each experiment was performed in quadruplicate). **Significantly different (P < 0.01) from non-specimen group using one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Concentration Assay

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 4. The cell-stimulating activity of ESG on TLR2-deficient mice PEC. PECs derived from TLR2-deficient and WT (BALB/c) mice (2 × 105 per 200 μL) were cultured with either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (TLR7 ligand; 5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/ mL). After 48 h, the culture fluid was withdrawn and the amounts of NO, IL-6 and MIP-2 in the supernatants were determined. White and black bars indicate the results for WT and TLR2KO PECs, respectively. Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. Values are the mean ± SD. Data are representative of three separate experiments (each experiment was performed in triplicate). ***Significantly different (P < 0.001) between WT and TLR2KO by an unpaired t-test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Derivative Assay, Cell Culture, Saline

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 6. The cell-stimulating activity of ESG toward TLR2/4DKO and MyD88KO PECs. PECs (2 × 105 per 200 μL) derived from TLR2/4DKO, MyD88KO and WT mice were incubated with either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/mL). After 48 h, the culture fluid was withdrawn and NO, IL-6 and MIP-2 in the supernatant was measured. The results of TLR2/4DKO and WT (C57BL/6) PECs are shown in (A)–(C), and the results of MyD88KO and WT (BALB/c) PECs are shown in (D)–(F). Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. Values are the mean ± SD. Data are representative of two independent experiments (each experiment was performed in triplicate). ***Significantly different (P < 0.001) between WT and KO mice by an unpaired t-test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Activity Assay, Derivative Assay, Incubation, Saline

Journal: Glycobiology

Article Title: Essential role of Toll-like receptor 2 in macrophage activation by glycogen.

doi: 10.1093/glycob/cwr122

Figure Lengend Snippet: Fig. 9. The binding activities of TLR2 to glycogen. (A) Bindings of ESG (100–1000 μg/mL) to TLR2 (2.5 μg/mL) were tested by ELISA. Pam3CSK4 (100 μg/mL), LPS (500 μg/mL) or imiquimod (500 μg/mL) was used as a control compound. Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. (B) Dose response binding curve at the indicated concentration of TLR2 to ESG (500 μg/mL). (C) Bindings of ESG to dectin-1 (β-glucan receptor) and comparison with other glucans such as oyster glycogen (natural-source glycogen; α-1,4/1,6 glucan), soluble starch (α-1,4/ 1,6 glucan), dextran (α-1,6 glucan), SPG (soluble β-1,3/1,6 glucan) and OX-CA (particle β-1,3/1,6 glucan). All glucans, Pam3CSK4, TLR2 and dectin-1 were tested with concentrations of 1000, 100, 2.5 and 1 μg/mL, respectively. OysGly and Pam3 in the graph indicate oyster glycogen and Pam3CSK4, respectively. Data are represented as the mean ± SD from three independent experiments (each experiment was performed in quadruplicate). **Significantly different (P < 0.01) from the negative control by one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Concentration Assay, Comparison, Starch, Negative Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress

doi: 10.1016/j.mcpro.2021.100069

Figure Lengend Snippet: Select basal and stress-induced interactors of OGT

Article Snippet: The following antibodies were used for Western blot analysis: OGT (O6264) and actin (ACTA1; A5060; MilliporeSigma), large proline-rich protein BAG6 (Bag6; A302-039A), coactivator-associated arginine methyltransferase 1 (CARM1) (A300-421A) and HCF1 (A301-400A; Bethyl, Montgomery, TX), heat shock cognate 71 (sc-7298), and TNKS1BP1 (sc-514517; Santa Cruz, Dallas, TX).

Techniques: Starch

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress

doi: 10.1016/j.mcpro.2021.100069

Figure Lengend Snippet: OGT interacts with effectors in a stress-dependent manner. A , validation of OGT interactions with known effectors. OGT was immunoprecipitated (IP: OGT) from the untreated (C), H 2 O 2 -treated (2.5 mM, 60 min, and 90 min), and OGT null (N) MEF lysate (2 mg total) followed by Western blot for OGT, CARM1, TNKS1BP, and BAG6. Inputs (10 μg) were also probed for HSC70 (n = 3). IgG indicates the contaminating signal arising from the AL24 immunoglobulin used to enrich OGT complexes. B , the presence of OGT and its interactors in immunoprecipitates were quantified by densitometry and normalized to the WT control. Error bars represent the SEM. The antibody used to detect TNKS1BP detects two species; only the upper species which runs at the molecular weight of TNKS1BP was quantified (Q>). Inputs and IPs were taken from the same gel, but different exposures. OGT interacts with known and novel interactors as validated by PRM. OGT was immunoprecipitated independently from untreated (WT), H 2 O 2 -treated (2.5 mM, 1.5 h, H 2 O 2 ), and null MEF cell lysates. After tryptic digestion, interactors were quantified using PRM: OGT ( C ), HCF1 ( D ), CARM1 ( E ), HDAC1 ( F ), SETD1A ( G ), GAPDH ( H ), and eIF3b ( I ). To normalize across immunoprecipitations, peptide intensities for each interactor were normalized to the intensities of two OGT peptides (AFLDSLPDVK and GSVAEAEDCYNTALR). Plotted WT ( blue ) and H 2 O 2 ( red ), D-I. Error bars represent the SEM. ∗ p ≤ 0.05. Bag6, large proline-rich protein BAG6; CARM1, coactivator-associated arginine methyltransferase 1; eIF3b, eukaryotic translation initiation factor 3 subunit B; H 2 O 2 , hydrogen peroxide; HCF1, host cell factor 1; HDAC1, histone deacetylase 1; HSC70, heat shock cognate 71; IgG, immunoglobulin G; MEF, mouse embryonic fibroblast; OGT, O-GlcNAc transferase; PRM, parallel reaction monitoring; SETD1A, histone-lysine N-methyltransferase SETD1A; TNKS1BP1, 182-kDa tankyrase-1–binding protein.

Article Snippet: The following antibodies were used for Western blot analysis: OGT (O6264) and actin (ACTA1; A5060; MilliporeSigma), large proline-rich protein BAG6 (Bag6; A302-039A), coactivator-associated arginine methyltransferase 1 (CARM1) (A300-421A) and HCF1 (A301-400A; Bethyl, Montgomery, TX), heat shock cognate 71 (sc-7298), and TNKS1BP1 (sc-514517; Santa Cruz, Dallas, TX).

Techniques: Biomarker Discovery, Immunoprecipitation, Western Blot, Control, Molecular Weight, Histone Deacetylase Assay, Targeted Proteomics, Binding Assay