Journal: Journal of neurochemistry

Article Title: A novel function of TBK1 as a target of Cdon in oligodendrocyte differentiation and myelination.

doi: 10.1111/jnc.13882

Figure Lengend Snippet: Fig. 2 Identification of TBK1 as a protein interacting with cell adhesion molecule- related, down-regulated by oncogenes (Cdon) in oligodendrocytes (OLGs) during early differentiation (a) Cdon or Goat IgG antibodies were used for immunoprecipitation (IP) in day 1 differentiated primary OLG cultures. A silver-stained sodium dodecyl sulfate– polyacrylamide gel with protein markers (M), IP input, Cdon antibody IP, and Goat IgG control IP is shown. The indicated band TBK1 was excised and analyzed by mass spectrometry. Molecular weights in kDa’s are indicated on the left. (b) Protein sequences of TBK1. The peptides identified by mass spectrometry are shown highlighted in yellow, cover ~ 14% of the protein sequence. (c) Cdon was immunoprecipitated from primary OLG cell lysates (day 1 differentiated) and the precipitated complex was analyzed by western blotting using antibodies specific for Cdon and TBK1. The experiment was reproduced with three different batches of primary OLG cultures. JLP and cAbl were shown as positive and negative control for IP.

Article Snippet: Primary antibodies were from the following suppliers: mouse monoclonal antibody (mmAb) anti-MBP (SMI-99) from Chemicon (Temecula, CA, USA); Goat IgG, Alexa Fluor 488-, 594-, Cy5-, or horseradish peroxidase-conjugated secondary antibodies from Southern Biotechnology (Birmingham, AL, USA), Jackson Immunoresearch Laboratories (Cedarlane, Hornby, ON, Canada), Bio-Rad Canada, or Invitrogen (Burlington, ON, Canada); Akt, phospho-Akt-T308, phospho-Akt-S473, phospho-ERK, ERK, JLP, GAPDH, phospho-TBK1, and TBK1 rabbit polyclonal antibodies for western blot from Cell Signaling Technology (Whitby, ON, Canada); TBK1 antibody for immunostaining from Abcam (Toronto, ON, Canada); TrueBlot conformation specific secondary antibodies from Rockland Inc. (Limerick, PA, USA); Cdon goat polyclonal antibody from R&D (Minneapolis, MN, USA); Olig2 rabbit polyclonal antibody from EMD Millipore (Darmstadt, Germany); cAbl and a-tubulin mmAb from Sigma-Aldrich; actin antibody from Santacruz (Dallas, TX, USA); Invitrogen green fluorescent protein (GFP) antibody from Thermo Fisher Scientific Inc. MAG antibody was a gift from Dr. Peter E. Braun (Emeritus Professor in Department of Biochemistry of McGill University).

Techniques: Immunoprecipitation, Staining, Control, Mass Spectrometry, Sequencing, Western Blot, Negative Control

Journal: Journal of neurochemistry

Article Title: A novel function of TBK1 as a target of Cdon in oligodendrocyte differentiation and myelination.

doi: 10.1111/jnc.13882

Figure Lengend Snippet: Fig. 4 TBK1 siRNA decreases expression of myelin-specific markers during oligodendrocyte (OLG) differentiation (a) siRNA knockdown efficiency. Forty-eight hours after siRNA treatment cells were changed into differentiation medium for 24 h followed by protein extraction. Targeted siRNA reduced levels of TBK1 protein. Densitometric quantification of the signals is shown below. (b) Two myelin- specific proteins, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), were reduced after nucleofection with TBK1 siRNA treatment during OLG differentiation. Densitometric quantification of western blot bands is shown in (d) as a relative ratio to GAPDH expression, which was used as a loading control in both (a) and (b). (c) Representative images of non- targeted Control and TBK1 siRNA- transfected cells. GalC is shown in red. MAG in green, and Olig2 in purple. (e) Quantification of the numbers of Olig2- positive cells expressing GalC or MAG. The values represent mean SEM of triplicate samples. Statistical differences were computed using independent t-tests (*p < 0.05, **p < 0.01, ***p < 0.005).

Article Snippet: Primary antibodies were from the following suppliers: mouse monoclonal antibody (mmAb) anti-MBP (SMI-99) from Chemicon (Temecula, CA, USA); Goat IgG, Alexa Fluor 488-, 594-, Cy5-, or horseradish peroxidase-conjugated secondary antibodies from Southern Biotechnology (Birmingham, AL, USA), Jackson Immunoresearch Laboratories (Cedarlane, Hornby, ON, Canada), Bio-Rad Canada, or Invitrogen (Burlington, ON, Canada); Akt, phospho-Akt-T308, phospho-Akt-S473, phospho-ERK, ERK, JLP, GAPDH, phospho-TBK1, and TBK1 rabbit polyclonal antibodies for western blot from Cell Signaling Technology (Whitby, ON, Canada); TBK1 antibody for immunostaining from Abcam (Toronto, ON, Canada); TrueBlot conformation specific secondary antibodies from Rockland Inc. (Limerick, PA, USA); Cdon goat polyclonal antibody from R&D (Minneapolis, MN, USA); Olig2 rabbit polyclonal antibody from EMD Millipore (Darmstadt, Germany); cAbl and a-tubulin mmAb from Sigma-Aldrich; actin antibody from Santacruz (Dallas, TX, USA); Invitrogen green fluorescent protein (GFP) antibody from Thermo Fisher Scientific Inc. MAG antibody was a gift from Dr. Peter E. Braun (Emeritus Professor in Department of Biochemistry of McGill University).

Techniques: Expressing, Knockdown, Protein Extraction, Western Blot, Control, Transfection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: TLR4 signalling: the key to controlling EV71 replication and inflammatory response

doi: 10.3389/fcimb.2024.1393680

Figure Lengend Snippet: EV71 infection inhibits the TLR4/MYD88/NF-κB and TBK1 pathway. (A) The network of TLR4 interacting proteins; (B) MYD88 gene expression of the GSE15323 dataset from the GEO database; (C–F) Western blot analysis of MYD88, p-NF-κB p65, NF-κB p65 and VP1 protein expression in EV71 infected RD cells at different MOIs. (G–I) Western blot analysis of p-TBK1/TBK1 and VP1 protein expression in EV71 infected RD cells at different MOIs. Data are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs control group.

Article Snippet: The membrane was then incubated with 5% skim milk powder at room temperature for 2 h, followed by incubation with specific antibodies including TLR4 (1:1000, WL00196, Wanleibio), NLRP3 (1:2000, WL02635, Wanleibio), COX2 (1:2000, WL01750, Wanleibio), IL-6 (1: 1000, WL02841, Wanleibio), IFN-γ (1:1000, WL02440, Wanleibio), β-actin (1:5000, BM0005, ABclonal), VP1 (1:1000, PAB7631-D01P, Abnova), TBK1 (1:1000, BM4038, Boster), p-TBK1 (1:1000, P00261, Boster) overnight at 4°C.

Techniques: Infection, Gene Expression, Western Blot, Expressing, Control