Journal: Molecular Medicine Reports

Article Title: Heme oxygenase 1‑overexpressing bone marrow mesenchymal stem cell‑derived exosomes suppress interleukin‑1 beta‑induced apoptosis and aging of nucleus pulposus cells

doi: 10.3892/mmr.2025.13481

Figure Lengend Snippet: Activation of the NF-κB signalling in NP cells induced by IL-1β was repressed after Exo-HO-1 treatment. Western blotting was performed to detect p-p65 and/or p65 protein levels in NP cells with different treatments (control, IL-1β, IL-1β + Exo and IL-1β + Exo-HO-1) as well as in the nuclear (N) and cytoplasmic (C) fractions of the cells. Histone H3 was used as an internal reference for the nucleus. ***P<0.001 vs. control; # P<0.05, ## P<0.01 and ### P<0.001 vs. IL-1β. NP, nucleus pulposus; IL, interleukin; Exo, exosomes; HO-1, heme oxygenase 1; p-phosphorylated.

Article Snippet: After blocking with a rapid blocking solution (Beyotime Institute of Biotechnology) at 4°C overnight, the membranes were incubated with appropriate primary antibodies including anti-HO-1 (cat. no. GTX637432; 1:2,000; GeneTex, Inc.), anti-TGS101 (cat. no. A01233-2, 0.25 μg/ml; Boster Bio), anti-CD63 (FNab01490; 1:1,000; Fine Biotech Co., Ltd, Wuhan, China) and anti-Cainexin (cat. no. GTX109669; 1:5,000; GeneTex, Inc.), Bcl-2 (cat. no. FNab00839; 1:1,000; Wuhan Fine Biotech Co., Ltd.), Bax (cat. no. FNab00810; 1:2,000; Wuhan Fine Biotech Co., Ltd.), cleaved caspase 3 (cat. no. MBS9410752; 1:1,000; MyBioSource, Inc.), anti-p65 (cat. no. PB9324; 0.5 μg/ml; Boster Bio), anti-p-p65 (cat. no. A00284S468-2; 1:2,000; Boster Bio), anti-GAPDH (cat. no. H00227; 1:5,000; Boster Bio) and anti-Histone H3 (cat. no. M12477-9; 1:1,000; Boster Bio) antibodies.

Techniques: Activation Assay, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Mechanisms of N‑acetylcysteine in reducing monocrotaline‑induced pulmonary hypertension in rats: Inhibiting the expression of Nox1 in pulmonary vascular smooth muscle cells.

doi: 10.3892/mmr.2017.7326

Figure Lengend Snippet: Figure 2. Expression of Nox 1 and Nox4. (A) The mRNA level of Nox1 and Nox4 in lung tissue. The standardization of group Cont was 1. #P<0.05 vs. Cont; *P<0.05 vs. MCT; **P<0.001 vs. MCT. (B) The mRNA level of Nox1 and Nox4 in PASMCs. The standardization of group Cont was 1. $P<0.05 vs. C1; +P<0.001 vs. M1. (C) Double immunofluorescence staining of Nox1 and α‑SMA in lung tissue. Green, α‑SMA; red, Nox1. (D) Western blotting and (E) densi tometric analysis of the expression of Nox1 protein in PASMCs (n=6). β‑actin was used as the internal control. $$P<0.001 vs. C1; ++P=0.011 vs. M1. (F) DHE fluorescence intensity of the lung tissue. (G) The standardization of group Cont was 1. The data demonstrate the fold change of group MCT and group NAC relative to group Cont. ##P<0.001 vs. Cont; **P<0.001 vs. MCT. (H) Correlation analysis of Nox1 and DHE fluorescence intensity. r=0.850; P<0.001. PASMC, pulmonary artery smooth muscle cell; Nox, NADPH oxidase; Cont, control; α‑SMA, smooth muscle actin; MCT, monocrotaline; NAC, N‑acetylcysteine; M, MCT‑treated; N, NAC‑treated group; C, control subgroup; DHE, dihydroethidium.

Article Snippet: Excess serum was absorbed and primary anti-Nox1 antibody (BA3720; 1:100; Boster Biological Technology) added for overnight incubation at 4 ̊C.

Techniques: Expressing, Double Immunofluorescence Staining, Western Blot, Control, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: Iron oxide magnetic nanoparticles combined with actein suppress non-small-cell lung cancer growth in a p53-dependent manner

doi: 10.2147/IJN.S127549

Figure Lengend Snippet: Fe 3 O 4 MNPs and AT induced apoptosis in NSCLC cells through caspase 3 activation. Notes: ( A ) Left: A549 cells were given AT, MNPs, or a combination of the two at the concentrations indicated for 24 hours. Total protein was extracted and subjected to immunoblot analysis for cleaved caspase 8, caspase 9, and caspase 3 detection. GAPDH was used as loading control. Right: quantification of Western blot analysis. ( B ) Left: H1975 cells were treated with AT, MNPs, or AT-MNP combination for 24 hours. Whole-cell protein was extracted and examined via immunoblot analysis for cleaved caspase 8, caspase 9 and caspase 3 determination. GAPDH was used as loading control. Right: quantification of Western blot analysis. ( C ) Caspase 3 (left) and caspase 9 (right) activation was determined in A549 cells administered AT and MNPs alone or a combination thereof in the absence or presence of caspase 3 or caspase 9 inhibitor for 24 hours. ( D ) Caspase 3 (left) and caspase 9 (right) activation was determined in H1975 cells treated with AT and MNPs alone or a combination thereof in the absence or presence of caspase 3 or caspase 9 inhibitor for 24 hours. Values are expressed as means ± standard error of mean. * P <0.05, ** P <0.01, and *** P <0.001 vs Con group; # P <0.05, ## P <0.01, and ### P <0.001. Analysis of variance and Dunnett’s analysis were included to compare the averages of multiple groups. Abbreviations: MNPs, magnetic nanoparticles; AT, actein; NSCLC, non-small-cell lung cancer; Con, control; NS, not significant.

Article Snippet: Rabbit anti-caspase 9 , 1:1,000 , Abcam (ab32539).

Techniques: Activation Assay, Western Blot

Journal: International Journal of Nanomedicine

Article Title: Iron oxide magnetic nanoparticles combined with actein suppress non-small-cell lung cancer growth in a p53-dependent manner

doi: 10.2147/IJN.S127549

Figure Lengend Snippet: Working model of the effect of Fe 3 O 4 magnetic nanoparticle and actein combination on NSCLC cells. Notes: Fe 3 O 4 magnetic nanoparticle and actein combination treatment induces apoptosis in NSCLC cells in a p53-dependent manner. p53 activation results in TRAIL and its DR4 and DR5 contributing to caspase 8 activity through FADD stimulation. Further, p53 activity reduces Bcl2 and BclXL expression, and promotes Bax and Bad expression levels. Therefore, the ratio of Bax:Bcl2 is increased, which leads to activation of caspase 9 and caspase 3. Consequently, apoptosis in NSCLC cells is induced, inhibiting NSCLC development. Abbreviation: NSCLC, non-small-cell lung cancer.

Article Snippet: Rabbit anti-caspase 9 , 1:1,000 , Abcam (ab32539).

Techniques: Activation Assay, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

doi: 10.1371/journal.pone.0129743

Figure Lengend Snippet: Primer Sequences for Real-time.

Article Snippet: The NOX1 and NOX4 antibodies were purchased from Boster Biological Technology Company, Wuhan, China.

Techniques:

Journal: PLoS ONE

Article Title: Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

doi: 10.1371/journal.pone.0129743

Figure Lengend Snippet: (A) HSC proliferation is reduced in NOX1KO and NOX4KO mice after CCl 4 injury. HSC proliferation in liver was presented by PCNA immunofluorescence microscopy. Desmin is a specific marker of HSCs (Red). PCNA is a marker of proliferation (Green). Original magnification X10. (B) Proliferation was reduced in HSCs lacking NOX1 and NOX4 in response to PDGF (10 ng/ml) for 24 h. Proliferative HSCs were presented by Ki67 (Red) immunofluorescence microscopy. Nuclei were presented by DAPI (Blue). Original magnification X10. (C) Graph of PCNA-positive HSCs in vivo. (D) Graph of Ki67-positive HSCs in vitro.

Article Snippet: The NOX1 and NOX4 antibodies were purchased from Boster Biological Technology Company, Wuhan, China.

Techniques: Immunofluorescence, Microscopy, Marker, In Vivo, In Vitro

Journal: PLoS ONE

Article Title: Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

doi: 10.1371/journal.pone.0129743

Figure Lengend Snippet: Human livers from controls (N = 7) and patients with cirrhosis (N = 10) were analyzed by Sirius Red staining and by immunofluorescence with antibodies against NOX1 and NOX4. The graphs show the percent positive area of the staining or immunofluorescence expressed as mean+S.D., p<0.05 for each comparison of control vs. fibrosis.

Article Snippet: The NOX1 and NOX4 antibodies were purchased from Boster Biological Technology Company, Wuhan, China.

Techniques: Staining, Immunofluorescence, Comparison, Control

Journal: The Journal of Clinical Investigation

Article Title: WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release

doi: 10.1172/JCI164222

Figure Lengend Snippet: ( A ) Genotyping of Wnk1 -cKO mice mediated by neuron-specific Syn1-Cre . Genomic tail-clip DNA was used for analysis. Lane 1, Wnk1 fl/+ ; Syn1-Cre ; lane 2, Wnk1 fl/fl ; lane 3, Wnk1 fl/fl ; Syn1-Cre . PCR is shown to detect WT vs. Wnk1 fl/fl locus (exon 2 and neo cassette are floxed). PCR forward primer F is located at exon 2. Reverse primers R1 and R2 are located at intron 2 and neo cassette, respectively. Note that Syn1-Cre is only active in neurons, so unexcised Wnk1 fl/fl locus is detected in tail-clip DNA. With large size neo cassette in the floxed locus, the F/R1 primer set does not amplify under the condition of PCR reaction. Additionally, PCR is shown to detect Syn1-Cre using Cre-specific primers. ( B ) Representative Western blot of WNK1 protein in WT and cKO brain regions shown relative to the kidney. Hippo, hippocampus. Cortex, cerebral cortex. ( C ) Quantitation (mean ± SEM) of 4 separate experiments, as shown in B . One WT and cKO mouse for each experiment. WNK1 was normalized to Gapdh and compared with the WT kidney (set as “1”). * P < 0.05, # P < 0.01, KO vs. WT by unpaired t test. ( D ) Atlas of brain section for immunofluorescent staining, as in E and F . OVLT (also known as vascular-organ-of-lamina-terminalis [VOLT]) is marked by a red line. 3V, third ventricle; MnPO, median preoptic nucleus; MPA, medial preoptic area. ( E and F ) Immunofluorescent staining of WNK1 in OVLT neurons colocalized with neuronal marker β-3 tubulin in WT ( E ) and cKO ( F ) mice. Scale bar: 100 μm.

Article Snippet: The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz).

Techniques: Western Blot, Quantitation Assay, Staining, Marker

Journal: The Journal of Clinical Investigation

Article Title: WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release

doi: 10.1172/JCI164222

Figure Lengend Snippet: ( A ) Water intake, ( B ) urine volume, ( C ) plasma osmolality, ( D ) urine osmolality, ( E ) plasma AVP level, and ( F ) copeptin level of control (Ctrl) and cKO mice at either ad libitum water intake or after 24-hour water restriction (WR). The inset in A shows Western blotting analysis of abundance of total and phospho-WNK1 (p-WNK1) using antibody against total WNK1 and against S382 phospho-WNK1. Arrowheads indicate molecular size 250 kDa. Lysates from WT OVLT tissue at ad libitum water intake and after 24-hour water restriction were immunoprecipitated by anti-WNK1 antibody and probed by anti-WNK1 and anti-p-WNK1 antibody. Representative of 4 separate experiments. Each experiment consists of 1 mouse ad libitum and 1 mouse on water restriction. For statistical analysis was performed with 2-way repeated ANOVA with Šidák post hoc analysis; for statistical analysis of the inset in A , unpaired 2-tailed t test was performed. For bar graphs, n = 6–8 mice, as indicated in scatter plots.

Article Snippet: The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz).

Techniques: Control, Western Blot, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release

doi: 10.1172/JCI164222

Figure Lengend Snippet: ( A ) Plasma osmolality, ( B ) [Na + ], ( C ) relative p-WNK1/WNK1 ratio in OVLT, ( D ) plasma AVP, ( E ) urine volume, ( F ) urine osmolality, and ( G ) water intake in WT and Wnk1 -cKO mice after mannitol or vehicle injection. Urine volume and water intake were measured 120 minutes after injection. Other measurements were taken 30 minutes after injection in separate mice from those in which urine and water intake were measured. The inset in C is representative of 3 experiments. Each experiment consists of 1 mouse injected with vehicle and 1 mouse injected with mannitol. Statistical analysis in A , B , and D was performed with 2-way repeated ANOVA with Šidák post hoc analysis; otherwise, unpaired t test was used. For bar graphs in A , B , D – G , n = 5 mice for each experimental condition, as indicated in scatter plot.

Article Snippet: The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz).

Techniques: Injection

Journal: The Journal of Clinical Investigation

Article Title: WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release

doi: 10.1172/JCI164222

Figure Lengend Snippet: ( A ) Ruptured whole-cell current-clamp recording for membrane potentials. Pipette and bath solution are indicated. ( B and C ) Membrane potentials of freshly isolated OVLT neurons at baseline, after incubation with 5 mM NaCl for 3 minutes and 5 minutes after washout of 5 mM NaCl hypertonicity. 600 pA currents were injected to depolarize membrane potential from the resting potential –55 mV to +150 mV. B and C represent examples of NaCl-responsive and nonresponsive neurons, respectively. ( D ) Treatment with pan-WNK kinase inhibitor (WNK463). Green and cyan bars indicate responsive (R) and nonresponsive (NR), respectively. WNK463 treatment significantly decreased the percentage distribution of responsive neurons vs. vehicle (Veh) treatment. P < 0.01, WNK463 vs. Veh, by 2-tailed Fisher’s exact test. ( E ) Wnk1 -cKO eliminated NaCl responsiveness. P < 0.01, cKO vs. WT, by 2-tailed Fisher’s exact test. In D and E , OVLT neurons were isolated form 4–5 mice for vehicle-treated, WNK463-treated, WT, and cKO groups.

Article Snippet: The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz).

Techniques: Membrane, Transferring, Isolation, Incubation, Injection

Journal: The Journal of Clinical Investigation

Article Title: WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release

doi: 10.1172/JCI164222

Figure Lengend Snippet: ( A ) Injection of AAV-retro-Cre virus into PVN of tdTomato-EGFP reporter mice resulted in green fluorescence in neurons of OVLT nuclei, which otherwise exhibited tomato red fluorescence. Scale bar: 200 μm. ( B ) PVN injection of AAV-retro-Cre virus into Wnk1 fl/fl mice resulted in deletion of Wnk1 in OVLT compared with control experiments with injection of AAV-retro-Cre virus into PVN of WT mice. Scale bar: 100 μm. ( C ) Urine volume, ( D ) urine osmolality, and ( E ) plasma osmolality of Wnk1 fl/fl mice before and after injection with AAV-retro-Cre virus during at libitum and after water restriction (WR). ( F ) Urine volume, ( G ) urine osmolality, and ( H ) plasma osmolality of WT mice before and after injection with AAV-retro-Cre virus. Data shown are mean ± SEM from before injection (labeled retro-AAV –) and after injection (labeled retro-AAV +). Statistical analysis by 2-way repeated ANOVA with Šidák post hoc analysis. n = 4–6 mice as indicated by scatter plots.

Article Snippet: The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz).

Techniques: Injection, Virus, Fluorescence, Control, Labeling

Journal: The Journal of Clinical Investigation

Article Title: WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release

doi: 10.1172/JCI164222

Figure Lengend Snippet: ( A and B ) Cooopeptin release in Wnk1 fl/fl and control WT mice with PVN injected with AAV-retro-Cre virus. Statistical comparison was made by paired t test between ad libitum and WR. ( C and D ) In separate groups of experimental ( Wnk1 fl/fl ) and control (WT) mice, OVLT neurons were isolated for recording of membrane potential oscillation. Pie charts show distribution of neurons that exhibit membrane potential oscillation responsive and nonresponsive to HTS (5 mM NaCl). P < 0.01 between pie chart in C and D by 2-tailed Fisher’s exact test. In A and B , n = 5 Wnk1 fl/fl and WT mice per experiment, as indicated in scatter plots.

Article Snippet: The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz).

Techniques: Control, Injection, Virus, Comparison, Isolation, Membrane

Journal: The Journal of Clinical Investigation

Article Title: WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release

doi: 10.1172/JCI164222

Figure Lengend Snippet: WT mice or mice heterozygous for GOF Cl – -insensitive Wnk1 -knockin ( Wnk1 -KI) allele received AAV-Cre virus injection in OVLT. ( A ) Relative abundance of phospho-OSR/SPAK (p-OSR/SPAK) in KI mice before (–) and after (+) injection, as measured by Western blotting analysis of OVLT using antibody against S373-phospho-SPAK/S325-phospho-OSR1. The inset shows representative Western blotting of 3 separate experiments. Each experiment consists of 3 replicates of WT and 3 Wnk1 -KI mice. Each data point in the bar graph represents the average of 3 replicates. Statistical analysis by unpaired t test. ( B ) Plasma AVP level, ( C ) urine volume, ( D ) urine osmolality in heterozygous Wnk1 -KI mice in which OVLT was injected with AAV-Cre virus, ( E ) plasma AVP level, ( F ) urine volume, and ( G ) urine osmolality of WT mice in which OVLT was injected with AAV-Cre virus. In B – G , n = 5 mice, as indicated in line plots. Statistical analysis by paired t test.

Article Snippet: The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz).

Techniques: Knock-In, Virus, Injection, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release

doi: 10.1172/JCI164222

Figure Lengend Snippet: ( A ) Schematic of the virus-mediated KO of Wnk1 in PVN-projecting OVLT neurons via injection of Cre-expressing retrograde virus at the PVN region. ( B ) Representative coronal section of the mouse brain injected with Cre-expressing virus at the PVN region. Scale bar: 1 mm. ( C ) Overlay of epifluorescence and IR-DIC images showing Cre-expressing neurons in the OVLT region. Scale bar: 10 μm. A recording pipette attached to a Cre-expressing cell was illustrated. ( D ) Top: Representative traces of spontaneous firing recorded from a NaCl-R neuron (R; cyan trace) and a NaCl-NR neuron (NR; red trace) in the WT mice. Bottom: Histogram of z score from the representative NaCl-R and NaCl-NR cells. ( E ) Distribution of the Δ z score in response to 5 mM NaCl stimulation of all recorded neurons in WT mice. The dashed line indicates 0.5. ( F ) Pie chart showing distribution of NaCl-R and NaCl-NR PVN-projecting OVLT neurons in WT mice. ( G ) Top: Representative traces of spontaneous firing recorded from a NaCl-R neuron and a NaCl-NR neuron in the Wnk1 –conditional KO (cKO) mice. Bottom: Histogram of z score from the representative NaCl-R and NaCl-NR cells. ( H ) Distribution of the Δ z score in response to 5 mM NaCl stimulation of all recorded neurons in Wnk1 -cKO mice. The dashed line indicates 0.5. ( I ) Pie chart showing distribution of NaCl-R and NaCl-NR PVN-projecting OVLT neurons in Wnk1 -cKO mice. * P = 0.032, between F and I , 2-tailed Fisher’s exact test. The WT group consists of recordings of 22 cells from 13 mice; the cKO group consists of 24 cells from 11 mice.

Article Snippet: The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz).

Techniques: Virus, Injection, Expressing, Transferring

Journal: The Journal of Clinical Investigation

Article Title: WNK1 promotes water homeostasis by acting as a central osmolality sensor for arginine vasopressin release

doi: 10.1172/JCI164222

Figure Lengend Snippet: ( A ) WNK1 exists in conformational equilibrium between chloride-bound autoinhibited dimer and chloride-free activation-competent monomer. Hyperosmolality extracts water from the cell and from the catalytic core of WNK1, which facilitates chloride unbinding, allowing autophosphorylation at S382 and be activated ( – ). WNK1 may activate Kv3.1 directly or indirectly through other intermediaries such as OSR1/SPAK. ( B ) Kv3.1 is a high-threshold voltage-gated K + channel activated by membrane depolarization to –20 mV or above ( , ). Activation of Kv3.1 shortens action potential duration, increases after hyperpolarization (AHP), and thus increases firing frequency (illustrated by red trace). Conversely, inhibition of Kv3.1 decreases firing frequency (blue trace). In support of this notion, we have found that TEA increased the action potential half-width (data not shown). ( C ) Exponential curvilinear relationship between AVP release and plasma osmolality begins at the threshold of approximately 280 mOsm/kg. WNK1 activation by cellular dehydration (Excitatory pathway; thick green line) plays an important role in AVP release by hyperosmolality. Additional mechanism(s) may be involved, at least for secretion at the basal state, which may include tonic inhibition of osmosensory neurons (Inhibitory pathway; thick solid red line). Loss of hypotonicity-mediated inhibitory pathway (thick dotted red line) may also contribute to hyperosmolality-induced AVP release. Compensation by the additional pathways may account for apparent similar AVP release defects in OVLT-selective deletion of WNK1 (by direct shRNA injection) versus neuronal deletion of WNK1. Extracellular hypertonicity may also activate WNK1 signaling cascade through molecular crowding of the protein (ref. ) (data not shown).

Article Snippet: The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz).

Techniques: Activation Assay, Membrane, Inhibition, shRNA, Injection

Journal: Scientific Reports

Article Title: Promotion of the inflammatory response in mid colon of complement component 3 knockout mice

doi: 10.1038/s41598-022-05708-8

Figure Lengend Snippet: Expression levels of members in the NF-κB signaling pathway. ( A ) Expression levels of NF-κB-p65 and IκB-α proteins were determined by Western blot analysis using specific primary antibody and HRP-labeled anti-rabbit IgG antibody. ( B ) Band intensities were determined using an imaging densitometer, and protein expressions were calculated relative to the intensity of β-actin. Tissue samples were collected from 3 to 5 mice per group, and each lysate was analyzed in duplicate for Western blot (final n = 6–10). Data are reported as the mean ± SD. * indicates p < 0.05 compared to the WT mice.

Article Snippet: Proteins (30 μg) were then separated by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h, and subsequently transferred to nitrocellulose membranes for 3 h at 40 V. Each membrane was then incubated separately, overnight at 4 °C, with the following primary antibodies: anti-C3 (ab200999, Abcam Com.), anti-C3aR (bs-2955R, Bioss Inc.), anti-CR1 (LS-C777464, LSBio Inc.), anti-COX-2 (12282, Cell Signaling Technology Inc., Danvers, MA, USA), anti-NLRP3 (15101, Cell Signaling Technology Inc.), anti-Cas 1 (24232, Cell Signaling Technology Inc.), anti-ASC (67824, Cell Signaling Technology Inc.), anti-iNOS (PA3-0304, Thermo Fisher Scientific Inc.), anti-ERK1/2 (9102, Cell Signaling Technology Inc.), anti-p-ERK (E-4) (9101, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-JNK (9252, Cell Signaling Technology Inc.), anti-p-JNK (9251, Cell Signaling Technology Inc.), anti-p38 (9212, Cell Signaling Technology Inc.), anti-p-p38 (9211, Cell Signaling Technology Inc.), anti-p-NF-κB-p65 (A00284T254, Boster Bio Inc., Pleasanton, CA, USA), anti-IκB (9242, Cell Signaling Technology Inc.), anti-p-IκB (9246S, Cell Signaling Technology Inc.), anti-IL-6 (SC-1265, Santa Cruz Biotechnology Inc.), anti-C5 (ab11898, Abcam Com.), anti-thrombin (ab92621, Abcam Com.), anti-E-cadherin (24E10, Cell Signaling Technology Inc.) or anti-β-actin (4967, Sigma-Aldrich Co.).

Techniques: Expressing, Western Blot, Labeling, Imaging