Journal: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: Schematic representation of genetic constructs CMV—CMV promoter: ( A ) wt PLOD2 ( LH2a ) expression plasmid, ( B ) wt PLOD2 ( LH2b ) expression plasmid, and ( C ) mutant Thr629Ala allele PLOD2 expression plasmid.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Construct, Expressing, Plasmid Preparation, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: Transfection efficiency of a plasmid with a GFP reporter. Flow cytometry analysis of transfection efficacy of HEK293 ( left ) and DF2 ( right ) cells in 24 h ( A ). Visualization of HEK293 ( left ) and DF2 ( right ) cells in 48 h using the EVOS FL Auto Imaging System ( B ). Green – PLOD2 fused to GFP. Scale bar—200 µm.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: Confocal microscopy images of HEK293 cells transfected with three different constructs, including GFP-LH2a ( B ), GFP-LH2b ( C ), and GFP-LH2b with mutation Thr629Ala ( D ) and wild-type without constructs ( A ). In Figure ( D ), the arrow shows a change in the morphology of the cell with expression of the LH2b (Thr629Ala). The left column (green) shows the images of GFP-tagged protein PLOD2, the middle column (red) shows the images of ER, and the right column shows the overlaid images of the left and middle columns. Scale bar—25 µm.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Confocal Microscopy, Transfection, Construct, Mutagenesis, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: List of primers used in this study.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Sequencing, Amplification, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: Cloning scheme.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Clone Assay, Construct

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Diagram of coronal rat brain section showing the site of injury and photographed region (4 A). Representative immunofluorescence images (10 ×) of ipsilateral cortex GFAP, ED-1, and HMGB1 immunoreactivity (green) combined with DAPI (blue) at 14 days post-TBI (10 days post-alcohol exposure) (4 B).

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Immunofluorescence

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Quantification of ipsilateral cortex GFAP, ED-1, and HMGB1 immunoreactivity at 14 days post-TBI (10 days post-alcohol exposure). Images are quantified as % area of positive staining in 1.035 mm2 (10×). Values are shown as means ± SEM. For GFAP and ED-1, Sham/Air n = 4, Sham/Alcohol n = 4, TBI/Air n = 4, TBI/Alcohol n = 6. For HMGB1, Sham/Air n = 4, Sham/Alcohol n = 5, TBI/Air n = 5, TBI/Alcohol n = 7 (each n represents the average of 3 images taken from each animal) * p < 0.05 of the TBI groups vs. time-matched sham controls; $ p < 0.05 of the TBI/Alcohol group vs. TBI/Air group, by two-way ANOVA (5 A, 5 C, and 5 E).

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Staining

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Primary and Secondary Antibodies.

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: A novel sodium signaling complex regulates uterine activity

doi: 10.1101/2020.07.31.229138

Figure Lengend Snippet: During the quiescent state, progesterone binding to the progesterone receptor (PR A/B ) increases NALCN expression and activity . Sodium current through NALCN activates SLO2.1 channels, increasing K + efflux to maintain the cell in a hyperpolarized state. As a result, voltage-dependent Ca 2+ channels (VDCCs) are closed, and uterine contractions do not occur. In the contractile state, estrogen acting on ERα inhibits NALCN expression , leading to decreased SLO2.1 activity. The reduced K + efflux depolarizes the membrane, leading to VDCC activation, an increase in intracellular Ca 2+ , and uterine contractility. At labor, Oxytocin (OXT) binds to the oxytocin receptor (OTR), leading to activation of phospholipase C (PLC), production of phosphatidylinositol 4,5-bisphosphate (PIP 2 ), and production of inositol triphosphate (IP 3 ). IP 3 activates the release of Ca 2+ from intracellular stores, and PIP 2 activates protein kinase C (PKC), which inhibits SLO2.1 . This SLO2.1 inhibition further depolarizes the membrane, thus opening more VDCCs, increasing intracellular Ca 2+ , and further activating myosin to cause muscle contraction.

Article Snippet: Duolink in situ proximity ligation assay (Sigma, St. Louis, MO) labeling was performed with the following antibodies: NALCN (mouse monoclonal, 1:100, StressMarq) and SLO2.1 (rabbit polyclonal, 1:200, Alomone).

Techniques: Binding Assay, Expressing, Activity Assay, Activation Assay, Inhibition

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Cellular Tight Junctions Prevent Effective Campylobacter jejuni Invasion and Inflammatory Barrier Disruption Promoting Bacterial Invasion from Lateral Membrane in Polarized Intestinal Epithelial Cells

doi: 10.3389/fcimb.2018.00015

Figure Lengend Snippet: TJ disruption is tightly associated with lipid raft-mediated C. jejuni invasion in polarized epithelial cells. (A–F) Caco-2 cells cultured for 7 days on transwells and treated with 4 mM EGTA for 20 min. Following EGTA treatment, cells were incubated in normal culture medium. During the incubation, the TER value (A) and ZO-1 localization (B–F) were evaluated every hour for 3 h. TER values were calculated as the percentage of the TER value for untreated cells. Scale bar = 10 μm. (G) Caco-2 cells treated with 4 mM EGTA for 20 min were incubated in normal culture medium for the indicated time. After incubation, cells were pretreated with 10 mM MβCD for 1 h and infected with C. jejuni for 6 h. The number of intracellular bacteria was measured using a gentamycin protection assay. Results are shown as the mean ± SD; n = 4. Significant difference from the post Ca 2+ reintroduction 0 h group are shown: N.S.; not significant; * P < 0.05; ** P < 0.01.

Article Snippet: Antibodies to the following were diluted in 3% BSA and used for Immunofluorescence staining ZO-1 (1:200; BD Bioscience), Alexa Fluor 568 (1:200, Molecular Probes).

Techniques: Cell Culture, Incubation, Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Cellular Tight Junctions Prevent Effective Campylobacter jejuni Invasion and Inflammatory Barrier Disruption Promoting Bacterial Invasion from Lateral Membrane in Polarized Intestinal Epithelial Cells

doi: 10.3389/fcimb.2018.00015

Figure Lengend Snippet: C. jejuni invasion is increased by active inflammation in polarized epithelial cells. (A–C) Caco-2 cells were treated with 20 ng/ml TNF-α for 48 h. Following TNF-α treatment, TER values (A) and ZO-1 localization (B,C) were evaluated. Scale bar = 10 μm. (D) Caco-2 cells were treated with 20 ng/ml TNF-α followed by treatment with 10 mM MβCD for 1 h and were infected with C. jejuni for 6 h. The number of intracellular bacteria was measured by gentamycin protection assay. Results are shown as the mean ± SD n = 4. Significant difference from the control or EGTA treated group are shown: N.S.; not significant; ** P < 0.01.

Article Snippet: Antibodies to the following were diluted in 3% BSA and used for Immunofluorescence staining ZO-1 (1:200; BD Bioscience), Alexa Fluor 568 (1:200, Molecular Probes).

Techniques: Infection