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: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Protective effects of Rosavin on bleomycin-induced pulmonary fibrosis via suppressing fibrotic and inflammatory signaling pathways in mice.

doi: 10.1016/j.biopha.2019.108870

Figure Lengend Snippet: Fig. 8. The effect of Rosavin on the expression of TGF-β1, α-SMA, Nrf2 and NF-κB p65 of BIM-induced PF in mice. Values are the mean ± SD (n = 3); nsP > 0.05, ##P < 0.01 vs. control group. *P < 0.05, **P < 0.01 vs. Bleomycin control group.

Article Snippet: Rabbit anti-TGF-β1 antibody (21898-1-AP) was purchased from Proteintech (Wuhan, China), and mouse anti-α-SMA antibody (BM0002), rabbit anti-NF-Kb p65 antibody, rabbit anti-Nrf2 antibody were all obtained from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: The N-Acetyl Phenylalanine Glucosamine Derivative Attenuates the Inflammatory/Catabolic Environment in a Chondrocyte-Synoviocyte Co-Culture System

doi: 10.1038/s41598-019-49188-9

Figure Lengend Snippet: IL-1β increases IKKα nuclear translocation and phosphorylation of serine 10 on histone H3, while NAPA attenuates these signalling events. ( A ) Overlapping signals of nuclear counterstaining (DAPI) and IKKα detected via an Alexa Fluor 555 secondary antibody: the colocalized signals indicate that NAPA addition is effective in reducing nuclear translocation of IKKα. ( B ) Upper pictures: left, western blot of anti-phosphorylated serine 10 of histone H3, along with GAPDH as a loading control and right: densitometric analysis of the signal showing the different pattern of H3pSer10 accumulation in CTR (circle), IL-1β (square) or IL-1β + NAPA (triangle) conditions. Lower images: specificity of the signal obtained with the anti-H3 phosphorylated serine 10: 20x field pictures of chondrocytes grown on coverslips in the bottom of wells at time 0 in control (upper row) or IL-1β stimulated conditions (lower row): Green: IKKα detected with an Alexa Fluor 488 anti-rabbit antibody; red: H3pSer10 signal detected with an Alexa Fluor 555 anti-mouse antibody; blue: nuclear DNA stained with Hoechst 33342 and merged images.

Article Snippet: IKKα staining was performed with 5 μg/ml rabbit anti-IKKα antibody (BOSTER Biological Technology, Freemont, CA, USA), while the extent of phosphorylation of serine 10 of histone H3 was evaluated with 5 μg/ml anti-phospho-histone H3, (Ser10), (mouse monoclonal, Upstate–Millipore) overnight at 4 °C.

Techniques: Translocation Assay, Phospho-proteomics, Western Blot, Control, Staining