Journal: Cells

Article Title: Protective Effects of SIRT6 Overexpression against DSS-Induced Colitis in Mice

doi: 10.3390/cells9061513

Figure Lengend Snippet: SIRT6 regulated expression and phosphorylation of TAK1. ( a ) The expression and phosphorylation levels of TAK1 were determined by immunohistochemistry. ( b , c ) The statistical results of ( a ). n = 3–6, ** p < 0.01.

Article Snippet: Sections then were blocked in 5% rat serum and incubated overnight at 4 °C with the diluted biotinylated primary antibody (1:500; rat-anti-mouse; anti-TAK1, #ab109526, Abcam, Cambridge, England, UK; anti-p-TAK1, #4508, Cell Signaling Technology, Danvers, MA, USA; anti-c-Jun, #ab40766, Abcam; anti-p-Jun, #ab32385, Abcam; anti-NF-κB p65 antibody, #ab32536, Abcam; anti-NF-κB p65 (phosphor S536), #ab86299, Abcam; anti-NF-κB p65 (acetyl K310) antibody, #ab19870, Abcam).

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: PDZ and LIM domain protein 2 plays dual and context-dependent roles in breast cancer development

doi: 10.1101/2020.01.27.920199

Figure Lengend Snippet: (A) Effect of EMT induction by TGFβ1 treatment (1 ng/ml for 24 hours) and by long-term hypoxia (96 h) on PDLIM2 protein levels. Successful EMT induction was confirmed by changes in the EMT markers E-cadherin (E-cad.), keratin-18 (KRT18) and β-catenin. (B) Effect of short-term hypoxia (48 and 72 hours) on PDLIM2 protein levels. Carbonic anhydrase-9 (CA9) was monitored as a control marker of hypoxia induction. (C) Effect of PDLIM2 overexpression on CA9 protein levels. (D) Effect of PDLIM2 protein level modulations on p53 protein levels, p53 (S20) phosphorylation (p-p53 S20), and on p65. Proliferating cell nuclear antigen (PCNA) was used as a loading control. Numbers under the protein bands represent their integral optical density (INT*mm 2 ). Blots are representative of two independent experiments (biological replicates), see Additional files 4 - 7: Figures S4 - S7 for both biological replicates.

Article Snippet: Mouse anti-E-cadherin (diluted 1:100) antibody (Ab) and anti-vimentin Ab (1:1000) were purchased from DakoCytomation, mouse anti-p65 Ab (1:500) was purchased from Santa Cruz Biotechnology, mouse anti-N-cadherin Ab was purchased from Invitrogen, mouse anti-actin Ab (1:250) was purchased from Sigma Aldrich and mouse anti-PDLIM2 Ab (1:250) was purchased from Origene.

Techniques: Control, Marker, Over Expression, Phospho-proteomics

Journal: bioRxiv

Article Title: PDZ and LIM domain protein 2 plays dual and context-dependent roles in breast cancer development

doi: 10.1101/2020.01.27.920199

Figure Lengend Snippet: (A) Effects of PDLIM2 protein level modulations on the EMT markers E-cadherin (E-cad.), N-cadherin (N-cad.) and vimentin (Vim.) as well as FAK levels, p53, p53 (S20) phosphorylation and p65. (B) Effects of EMT induction by treatment with TGFβl (1 ng/ml for 24 hours) on PDLIM2 protein levels and EMT markers. Numbers under the protein bands represent their integral optical density (INT*mm 2). Blots are representative of two independent experiments (biological replicates), see Additional files 8 - 9: Figures S8 - S9 for both biological replicates.

Article Snippet: Mouse anti-E-cadherin (diluted 1:100) antibody (Ab) and anti-vimentin Ab (1:1000) were purchased from DakoCytomation, mouse anti-p65 Ab (1:500) was purchased from Santa Cruz Biotechnology, mouse anti-N-cadherin Ab was purchased from Invitrogen, mouse anti-actin Ab (1:250) was purchased from Sigma Aldrich and mouse anti-PDLIM2 Ab (1:250) was purchased from Origene.

Techniques: Phospho-proteomics

Journal: PLoS ONE

Article Title: DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1 G93A model of amyotrophic lateral sclerosis in vivo and in vitro

doi: 10.1371/journal.pone.0183684

Figure Lengend Snippet: ( A-C’ ) Representative photomicrographs depicted in the ventral cervical spinal cord of control (A, A’) , presymptomatic (B, B’) and diseased (C, C’) hSOD1 G93A transgenic mice. In MNs (green), the cytoplasmic TDP-43 signal (red) did not exceed background levels, whereas the nuclear TDP-43 signal was strong in specimens of both control and hSOD1 G93A mice. Similar to in vivo conditions, cultured (D-E’) non-transgenic (D-D’) and transgenic (E, E’) MNs displayed a comparably low cytoplasmic TDP-43 signal, which was not increased in MNs carrying the hSOD1 G93A mutation. Nuclear TDP-43 location was verified by DAPI counterstaining (blue). (F) Representative western blot of TDP-43 in cytoplasmic (CP), soluble nuclear (sNE) and chromatin-bound nuclear (cNE) subcellular extracts derived from ventral cervical and thoracic spinal cords of control (ctrl), presymptomatic (PS) and diseased (DS) hSOD1 G93A mice. Under resting conditions, detection of cytoplasmic p65 (RelA) was utilized as a loading control and for purity validation of subcellular fractions. The cytoplasmic fraction was free of TDP-43, whereas TDP-43 was present in both nuclear extracts, with a higher abundance in the cNE than the sNE fraction. Scale bars depict 20 μm (A-C’) and 10 μm (D-E’).

Article Snippet: The following primary antibodies were used: rabbit anti-TDP-43 (1:2,500; polyclonal; Acris Antibodies, RRID: AB_615042), rabbit anti-NF-κB p65 (1:1,000; polyclonal; Santa Cruz Biotechnology, Dallas, TX, US, RRID: AB_632037) and mouse anti-NF-κB p65-active subunit (1:1,000; monoclonal; Merck Chemicals, RRID: AB_2178887).

Techniques: Transgenic Assay, In Vivo, Cell Culture, Mutagenesis, Western Blot, Derivative Assay

Journal: Cells

Article Title: Gas6/TAM Signalling Negatively Regulates Inflammatory Induction of GM-CSF in Mouse Brain Microglia

doi: 10.3390/cells10123281

Figure Lengend Snippet: Gas6 inhibits LPS-induced nuclear translocation of the NF-κB p65 subunit in microglia. Pure microglial cell cultures were treated with LPS (10 ng/mL) for 30 min with or without 1 h pre-incubation with Gas6 (1.6 µg/mL), which then remained throughout. ( A ) Cells underwent immunofluorescence staining with anti-p65 primary antibody with AlexaFluor647 anti-mouse secondary antibody and DAPI nuclear counterstaining. Scale bar = 100 µm. ( B ) p65 staining within the nuclear area of each cell was quantified for each treatment group. Data is shown in a violin plot with median and interquartile ranges visible ( n > 30 cells). Data was statistically analysed using one-way ANOVA; **** p < 0.0001 vs. both other conditions.

Article Snippet: For staining, coverslips were incubated in primary mouse anti-p65 antibody (1:300; Proteintech, Manchester, UK) at 4 °C overnight.

Techniques: Translocation Assay, Incubation, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Aryl Hydrocarbon Receptor Regulates Apoptosis and Inflammation in a Murine Model of Experimental Autoimmune Uveitis

doi: 10.3389/fimmu.2018.01713

Figure Lengend Snippet: Aryl hydrocarbon receptor (AhR) activation inhibited nuclear factor (NF-κB) and signal transducers and activators of transcription (STAT) signaling pathways in experimental autoimmune uveitis (EAU) mice. (A–C) Representative western blotting images of NF-κB p65, p-stat1, and p-stat3 in retinas of AhR −/− and AhR +/+ EAU mice and their quantifications ( n = 4/group; mean ± SD; ** p < 0.01; unpaired Student’s t -test). (D–F) Representative Western blotting images of NF-κB p65, p-stat1, and p-stat3 in retinas of naive, vehicle, and TCDD-treated mice and their quantifications ( n = 4/group; mean ± SD; *** p < 0.001; one-way ANOVA).

Article Snippet: Primary antibodies used included rabbit anti-CD16 (1:20,000, Abcam, UK), rabbit anti-CD206 (1:1,000, Abcam, UK), rabbit anti-iNOS (1:200, Proteintech, Wuhan, China), rabbit anti-arginase-1 (Arg-1) (1:200, Proteintech, Wuhan, China), rabbit anti-occludin (1:200, Proteintech, Wuhan, China), rabbit anti-claudin-5 (1:1,000, Abcam, UK), rabbit anti-zonula occludens-1 (ZO-1) (1:200, Proteintech, Wuhan, China), rabbit anti-cleaved caspase-3 (1:2,000, Abcam, UK), rabbit anti-Bcl-2 (1:500, Wanleibio, Liaoning, China), rabbit anti-Bcl-2 associated X protein (Bax) (1:1,000, Abcam, UK), mouse anti-p65 (1:500, Servicebio, Wuhan, China), rabbit anti-phospho-signal transducers and activators of transcription (STAT)1 (1:500, Cell Signaling Technology, MA, USA), rabbit anti-phospho-STAT3 (1:500, Servicebio, Wuhan, China), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (1:2,000, Proteintech, Wuhan, China) was used as a loading control.

Techniques: Activation Assay, Protein-Protein interactions, Western Blot

Journal: iScience

Article Title: ERK1-mediated immunomodulation of mesenchymal stem cells ameliorates inflammatory disorders

doi: 10.1016/j.isci.2023.107868

Figure Lengend Snippet:

Article Snippet: Anti- p -NFκB antibody , Cell signaling Technology , Cat # 3031S; RRID: AB_330559.

Techniques: Recombinant, Plasmid Preparation, Luciferase, Software