Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Changes in Elastic Moduli of Fibrin Hydrogels Within the Myogenic Range Alter Behavior of Murine C2C12 and Human C25 Myoblasts Differently

doi: 10.3389/fbioe.2022.836520

Figure Lengend Snippet: Primer sequences, primer concentrations and thermal profiles used for qPCR.

Article Snippet: Blocking was performed with PBS/T-1% bovine serum albumin (BSA) (w/v) at room temperature for 1 h. The primary antibody targeting all MHC isoforms (MF 20, Developmental Studies Hybridoma Bank, Iowa, United States, RRID: AB_2147781) was diluted 1:300 in PBS/T-1% BSA (w/v) and incubated overnight at 4°C.

Techniques:

Journal: Autophagy

Article Title: USP19 (ubiquitin specific peptidase 19) promotes TBK1 (TANK-binding kinase 1) degradation via chaperone-mediated autophagy

doi: 10.1080/15548627.2021.1963155

Figure Lengend Snippet: USP19 deficiency in macrophages enhanced VSV-induced type I interferon production and antiviral response by suppressing CMA-mediated TBK1 degradation. (A and B) qPCR analysis of Ifna4 (A) and Ifnb1 (B) expression in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated times with or without VER-155,008 (5 μM) pretreatment. (C and D) qPCR analysis of Ifna4 (C) and Ifnb1 (D) expression in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages transfected with poly(I:C) (1 μg/mL) for the indicated hours with or without pretreatment with VER-155,008 (5 μM). (E and F) qPCR analysis of Ccl5, Cxcl10 (E), Isg15, Ifit2, Ifit1, and Mx1 expression (F) in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for 12 h with or without pretreatment with VER-155,008 (5 μM). (G) Immunoblot analysis of OSA1, EIF2AK2, and LAMP2A expression in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated durations. (H and I) Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages were infected with VSV-GFP for 12 h with or without VER-155,008 (5 μM) pretreatment, and images were captured under a fluorescence microscope (H). The percentage of GFP+ cells was determined via flow cytometry (I). (J) qPCR analysis of VSV replication in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) with or without VER-155,008 (5 μM) pretreatment. (K) Determination of VSV titers in supernatants via a TCID50 assay of Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV with or without VER-155,008 (5 μM) treatment. (L) Immunoblot analysis of the indicated proteins in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated durations. (M) Immunoblot analysis of STAT1 and STAT2 phosphorylation in Usp19flox/flox and usp19flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated hours. The data are representative of three independent experiments. Scale bars: 100 μm. Error bars show the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student’s t test.

Article Snippet: Antibodies specific to p-IRF3 (4947), IRF3 (4302), p-TBK1 (5483), TBK1 (3504), p-STAT1 (9167), STAT1 (14,994), p-STAT2 (88,410), STAT2 (72,604), DDX58 (3743), and MAVS (4983) were purchased from Cell Signaling Technology.

Techniques: Expressing, Infection, Transfection, Western Blot, Fluorescence, Microscopy, Flow Cytometry, TCID50 Assay, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: A Single Amino Acid Mutation Converts ( R )-5-Diphosphomevalonate Decarboxylase into a Kinase *

doi: 10.1074/jbc.M116.752535

Figure Lengend Snippet: Radio-TLC analyses of the products of the wild type and mutants of SsoDMD (A) and TacM3K (B). The asterisk indicates an unknown product from the reaction with MVA-5-P and the D281N mutant, which seems distinct from unknown compounds synthesized by the reaction with MVA-5-PP and the D281T or D281V mutant. ori., origin; s.f., solvent front.

Article Snippet: [2- 14 C]MVA and [2- 14 C]MVA-5-PP were enzymatically synthesized from [2- 14 C]MVA-5-P using Antarctic phosphatase (New England Biolabs) and recombinant S. solfataricus PMK, respectively, as described in our previous work ( 28 ).

Techniques: Mutagenesis, Synthesized

Journal: The Journal of Biological Chemistry

Article Title: A Single Amino Acid Mutation Converts ( R )-5-Diphosphomevalonate Decarboxylase into a Kinase *

doi: 10.1074/jbc.M116.752535

Figure Lengend Snippet: 13C NMR analysis of the product of the D281T mutant. Parts of the 13C NMR spectra after reaction without (A, C, E, G, and I) or with (B, D, F, H, and J) the D281T mutant are shown. Green asterisks indicate the indistinguishable signals of [U-13C]MVA-5-P and [U-13C]MVA-5-PP, which are derived from the conversion of [U-13C]MVA by MVK and PMK from S. solfataricus. Black asterisks in A represent the signals of [U-13C]MVA-5-P, which are distinguishable from those of [U-13C]MVA-5-PP indicated by green asterisks. Red asterisks indicate the signal of the product of the D281T mutant. The spectra corresponding to the signals of C3 (A and B), C1 (C and D), C2 (E and F), C4 (G and H), and C6 (I and J) of MVA-5-PP and the new product are shown. The reaction with the D281V mutant gave almost the same results as those from the reaction with D281T (data not shown).

Article Snippet: [2- 14 C]MVA and [2- 14 C]MVA-5-PP were enzymatically synthesized from [2- 14 C]MVA-5-P using Antarctic phosphatase (New England Biolabs) and recombinant S. solfataricus PMK, respectively, as described in our previous work ( 28 ).

Techniques: Mutagenesis, Derivative Assay