Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 1. Average total fold intensity of phospho-protein signal as compared to median control value. Colored bars (dark blue = 0.5 Gy, light blue = 0.1 Gy and green = 0.05 Gy) indicate values significantly different from controls (yellow). Significance bars are shown for all significant differences between doses and 0 (p ≤0.05). Average fold intensity levels are shown at 2 h post radiation for γH2AX (A), pATF2 (B) and pSMC1 (C). Persistent effects are shown at 24 h for γH2AX (D), pATF2 (E) and pSMC1 (F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques: Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 3. Average total fold pATF2 intensity over median control level versus fluence for various radiation qualities. Average fold intensity levels are shown at 2 h post radiation for Si ions (A),Fe ions (C) and Ti ions (E). Persistent effects are shown at 24 h for Si ions (B), Fe ions (D) and Ti ions (F).

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques: Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 6. Average total fold intensity of pATF2 signal intensity divided by fluence and graphed versus LET. Average fold intensity levels are shown at 2 h post radiation for 0.05 Gy (A), 0.1 Gy (B) and 0.5 Gy (C). Persistent effects are shown at 24 h for 0.05 Gy (D), 0.1 Gy (E) and 0.5 Gy (F).

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing Ras-Related C3 Botulinum Toxin Substrate 1 Inhibits Growth and Migration of Hypopharyngeal Squamous Cell Carcinoma via the P38 Mitogen-Activated Protein Kinase Signaling Pathway

doi: 10.12659/MSM.907468

Figure Lengend Snippet: Silencing Rac1 inhibits the activation of P38 MAPK signal in vitro . The levels of MKK3 and p-MKK3 ( A, B ) and P38 and p-P38 ( C, D ) were detected by Western blot analysis. GAPDH served as the internal reference. All experiments were repeated 3 times and the results are presented as mean ±SD. *** P<0.001 compared with the negative control group.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin, and then incubated with Rac1 antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1: 1000; Proteintech, Wuhan, China), Cyclin D1 antibody (1: 1000; BOSTER, Wuhan, China), Cyclin E antibody, Cyclin B antibody (1: 500; Bioss, Beijing, China), metal matrix proteinase (MMP)-2 antibody, MMP-9 antibody, B cell lymphoma-2 (Bcl-2) antibody, Bcl-2-associated X protein (Bax) antibody, phosphorylated-MAP kinase (p-MKK3) antibody (1: 500; Sangon Biotech, Shanghai, China), caspase-3 antibody, caspase-9 antibody, poly ADP-ribose polymerase (PARP) antibody (1: 1000; Cell Signaling Technology, Beverly, MA, USA), p38 mitogen-activated protein kinase (MAPK) antibody, p-p38 MAPK antibody (1: 500; KeyGen, Nanjing, China), or MKK3 antibody (1: 300; BOSTER) overnight at 4°C.

Techniques: Activation Assay, In Vitro, Western Blot, Negative Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing Ras-Related C3 Botulinum Toxin Substrate 1 Inhibits Growth and Migration of Hypopharyngeal Squamous Cell Carcinoma via the P38 Mitogen-Activated Protein Kinase Signaling Pathway

doi: 10.12659/MSM.907468

Figure Lengend Snippet: Rac1 silencing inhibits the activation of P38 MAPK signal in vivo . The levels of MKK3 and p-MKK3 ( A, B ) and P38 and p-P38 ( C, D ) in each group were detected by Western blot analysis with GAPDH as the internal reference. All experiments were repeated 3 times. The results are presented as mean ±SD. N=6. *** P<0.001 compared with the negative control group.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin, and then incubated with Rac1 antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1: 1000; Proteintech, Wuhan, China), Cyclin D1 antibody (1: 1000; BOSTER, Wuhan, China), Cyclin E antibody, Cyclin B antibody (1: 500; Bioss, Beijing, China), metal matrix proteinase (MMP)-2 antibody, MMP-9 antibody, B cell lymphoma-2 (Bcl-2) antibody, Bcl-2-associated X protein (Bax) antibody, phosphorylated-MAP kinase (p-MKK3) antibody (1: 500; Sangon Biotech, Shanghai, China), caspase-3 antibody, caspase-9 antibody, poly ADP-ribose polymerase (PARP) antibody (1: 1000; Cell Signaling Technology, Beverly, MA, USA), p38 mitogen-activated protein kinase (MAPK) antibody, p-p38 MAPK antibody (1: 500; KeyGen, Nanjing, China), or MKK3 antibody (1: 300; BOSTER) overnight at 4°C.

Techniques: Activation Assay, In Vivo, Western Blot, Negative Control

Journal: eBioMedicine

Article Title: Preclinical characterisation of the protective capacity of an anti-nucleoprotein hRSV monoclonal antibody

doi: 10.1016/j.ebiom.2025.106104

Figure Lengend Snippet: The humanised anti-N-hRSV mAbs demonstrate antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity in vitro . A. The percentage of antibody-dependent cellular cytotoxicity (ADCC) activity for each clone of the humanised anti-N-hRSV mAbs (clones P1-04H, P1-05D, P2-01A, and P2-01D) was measured using luciferase expression levels in Jurkat NFAT-luc FcγRIII cells incubated with the humanised anti-N-hRSV mAbs against N-hRSV. B. The percentage of complement-dependent cytotoxicity (CDC) activity for each clone was assessed by viability assay of HEp-2 cells infected with hRSV-GFP, treated with the four clones of the humanised anti-N-hRSV mAbs, followed by the addition of mouse purified complement. Three independent experiments were conducted for each ADCC and CDC assay (N = 3).

Article Snippet: After 30 min of antibody incubation, 100 μl of a 1:25 solution of mouse-purified complement (Rockland Immunochemical; cat #R.C201-0005) was added, bringing the final concentration in the well to 1:50, and the plate was incubated for 3 h at 37 °C with 5% CO 2 .

Techniques: Activity Assay, In Vitro, Clone Assay, Luciferase, Expressing, Incubation, Viability Assay, Infection, Purification, CDC Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: SE-associated LINC01013 was transcriptionally activated by CEBPB in hPASMCs. A Prediction of candidate transcription factors and binding sites. (Transcription factor related databases: JASPAR: https://jaspar.elixir.no/ ; PROMO: https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 ; GENECARD: https://www.genecards.org/ ; AnimalTFDB: http://bioinfo.life.hust.edu.cn/HumanTFDB/#!/ ; Super enhancer related database: LncSEA: https://bio.liclab.net/LncSEA/ ). B The schematic diagram illustrates the LINC01013 promoter, divided into segments P1-P4, and the binding sites of transcription factors. C Four constituents (E1-E4) of SE region of LINC01013 derived from the WashU Epigenome Browser databases ( http://epigenomegateway.wustl.edu/browser/ ). D , E hPASMCs were subjected to ChIP analysis using antibodies against H3K27ac, H3K4me1 and CEBPB. The association with the SE region (D, E1-E4) and promoter region (E, P1-P4) of LINC01013 was quantified by RT‒qPCR ( n = 3). F hPASMCs were treated with CEBPB siRNA and subjected to ChIP analysis using antibodies against H3K27ac. The association with the E2 of SE (left) and P1-P3 promoter regions (right) of LINC01013 was quantified by RT-qPCR ( n = 3). G Schematic diagram of transcribing LINC01013 in hPASMCs. All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA or Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; IP, immunoprecipitation; IgG, Immunoglobulin G; TSS, transcription initiation site

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Binding Assay, Derivative Assay, Quantitative RT-PCR, Negative Control, Immunoprecipitation

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