Journal: bioRxiv

Article Title: MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS pathophysiology

doi: 10.1101/2023.01.23.524851

Figure Lengend Snippet: a,b) Quantification of nuclear pBrd4 levels by confocal IF with anti-pBrd4 (green) and DAPI staining of primary microglial cells pre-treated with 10 μM C13 (or DMSO) for 1 h and stimulated or not with 1 μg/mL LPS for 4 h. The data and images shown correspond to one experiment (N=20 analyzed images per condition) and are representative of two independent experiments. Scale bar: 10 μm. Student’s t-test (between two groups as indicated), unpaired, one-tailed. c,d) Quantification (c) and representative image (d) of Western blot analyses with anti-pBrd4 and anti-Brd4 (normalized to Brd4 or α-tubulin signal, respectively) in lysates of WT and MOK-KO SIM-A9 cells stimulated with 1 μg/mL LPS for 1 h. Data are from three independent experiments (N=3). Student’s t-test, unpaired, one-tailed. e,f) Quantification (e) and representative images (f) of confocal IF analyses of nuclear pBrd4 levels with anti-pBrd4 (green) and DAPI staining of WT and MOK-KO SIM-A9 cells stimulated or not with 1 μg/mL LPS for 1 h. Signal was enhanced by 50% in all four images in the first row for better visualization. Images are from one out of three independent experiments (N=3). Scale bar: 10 μm. Student’s t-test, unpaired, two-tailed. g) ChIP-PCR assessment of Brd4-binding to specific cytokine promoters. Represented data are relative values (percentage of input) of PCR products for IL-6, IFNβ and TNFα promoters after ChIP assay with either anti-Brd4 or IgG control antibodies with chromatin isolated from WT or MOK-KO cells treated or not with 1 μg/mL of LPS for 1 hour (N=4). Student’s t-test, paired, one-tailed. Data are the mean -/+ S.E.M. from ‘N’ independent experiments, and *P<0.05, **P<0.01 and ***P<0.001. NS: not significant.

Article Snippet: For intracellular labelling of bi-phosphorylated MOK protein (bpMOK), cells were fixed and permeabilized with the Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences, cat. 554722) according to the manufacturer instructions and stained with anti-MOK antibody bi-phosphorylated (pThr159+pTyr161, StressMarq, cat. SPC-1030), followed by donkey anti-rabbit IgG-Alexa Fluor 488 (Invitrogen, cat. A-21206).

Techniques: Staining, One-tailed Test, Western Blot, Two Tailed Test, Binding Assay, Control, Isolation