Journal: PLoS ONE

Article Title: Fibro-Vascular Coupling in the Control of Cochlear Blood Flow

doi: 10.1371/journal.pone.0020652

Figure Lengend Snippet: ( A ) Type V fibrocytes positive for S100 (green) abut capillary walls labeled by isolectin IB4 (blue). ( B ) Type V fibrocytes are positive for Na + /K + ATPase β1 (red). ( C ) Type V fibrocytes also contain high levels of NO, as detected with the intracellular NO indicator, DAF-2DA (gray). ( D ) Magnification of panel B shows foot processes in contact with a capillary. ( E ) A multiple-foot process of a fibrocyte abuts a capillary wall. ( F ) A high magnification image shows a fibrocyte end-foot structure at the soma of a pericyte. The somas of the pericytes were labeled by an antibody for NG2, (red), and processes were labeled with an antibody for the structural protein, desmin (blue).) Capillary walls are labeled by phalloidin (green). ( G ) and ( H ) Fibrocytes contact capillaries with enlarged endings. ( I ) The endings display electron-dense membrane regions rich in mitochondria. Abbreviations: FC, fibrocyte; EC, endothelial cells; PC, pericyte; Mt, mitochondria. Calibration bars in H and I are 500 nm.

Article Snippet: The primary antibodies used in the experiments included anti-desmin (rabbit monoclonal to desmin, cat# ab32362, Abcam, Cambridge, MA), anti-collagen type IV (cat# ab6586, Abcam, Cambridge, MA), anti-COX1 (cat# Sc-1752, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-S100 (cat# ab8330, Abcam, Cambridge, MA), and anti-Na + /K + ATPase β1 (cat# 06-170, Upstate, Lake Placid, NY).

Techniques: Labeling

Journal: Cardiovascular Diabetology

Article Title: Dysregulated Rbfox2 produces aberrant splicing of Ca V 1.2 calcium channel in diabetes-induced cardiac hypertrophy

doi: 10.1186/s12933-023-01894-5

Figure Lengend Snippet: Ca V 1.2 alternative exon 9* is specifically increased in diabetic heart . ( A ) The membrane expression of Ca V 1.2 α 1C was detected by Western blotting in heart tissues from control and HFD/STZ-treated rats, Na-K ATPase protein was detected as internal control. The relative band densities were analyzed and normalized to Na-K ATPase. n = 6 rats for each group. * P = 0.0166, unpaired t test. ( B ) Schematic diagram shows the locations of the PCR primers designed to amplify and detect rat Ca V 1.2 inclusive of or in the absence of alternative exons in cardiac tissues. Total RNA was extracted from hearts, and PCR products amplified from cDNA libraries were separated on 2.5% agarose gel. Actb mRNA was detected as internal control. Rat Cacna1c mRNAs with exon 8 or 8a were amplified by RT-PCR, followed by digestion with restriction endonuclease BamHI. The value for percent exon 8a inclusion were the lower 2 bands’ intensity divided by the sum of the intensities of upper and lower bands. n = 4 rats for each group. P = 0.4107, unpaired t test. ( C ) The value for percent exon 9* inclusion was the upper band intensity divided by the summed intensities of upper and lower bands. n = 6 rats for each group. **P < 0.0001, unpaired t test. ( D ) The value for percent exon 33 inclusion was also presented as a bar chart. n = 6 rats for each group. P = 0.4107, unpaired t test. ns indicates no significant differences

Article Snippet: Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca V 1.2 α 1C (1.6 μg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 μg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus).

Techniques: Membrane, Expressing, Western Blot, Control, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Cardiovascular Diabetology

Article Title: Dysregulated Rbfox2 produces aberrant splicing of Ca V 1.2 calcium channel in diabetes-induced cardiac hypertrophy

doi: 10.1186/s12933-023-01894-5

Figure Lengend Snippet: GS application induces hyperpolarized window currents of Ca V 1.2 channel in NRVMs . ( A ) NRVMs were treated with 10% NG or GS for 48 h, then the membrane protein was extracted. Ca V 1.2 α 1C subunit was detected, and Na-K ATPase was detected as internal control. ( B ) Relative expression of Ca V 1.2 α 1C subunit was normalized with Na-K ATPase. n = 3 independent experiments. P = 0.7251, unpaired t test. ns indicates no significant differences. ( C ) Ca V 1.2 channel currents were recorded from NRVMs with NG, GS or GS + Gö6983 treatment in 10 mmol/L Ba 2+ bath solution. ( D ) I-V relationship curves of Ca V 1.2 channel were recorded under different testing potentials in NRVMs. ( E ) Current densities of Ca V 1.2 channel in NRVMs treated with NG, GS or GS + Gö6983 were presented. ( F ) Plots of steady-state activation (SSA) curve of Ca V 1.2 channel were derived from I - V currents in differentially-treated NRVMs. ( G ) Plots of the steady-state inactivation (SSI) curve were recorded and analyzed in NRVMs. ( H ) Window currents were superimposed from SSI ( f ∞ ) and SSA ( d ∞ ) curves of NG, GS or GS + Gö6983-treated NRVMs

Article Snippet: Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca V 1.2 α 1C (1.6 μg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 μg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus).

Techniques: Membrane, Control, Expressing, Activation Assay, Derivative Assay

Journal: Cardiovascular Diabetology

Article Title: Dysregulated Rbfox2 produces aberrant splicing of Ca V 1.2 calcium channel in diabetes-induced cardiac hypertrophy

doi: 10.1186/s12933-023-01894-5

Figure Lengend Snippet: Knockdown of Rbfox2 hyperpolarizes window currents of Ca V 1.2 channel in NRVMs . ( A ) The protein expression of Rbfox2 and β-actin were detected in whole-cell lysate of isolated NRVMs by using Western blotting after transfecting with NT or Rbfox2 siRNAs. The membrane protein was also extracted, and membrane expression of Ca V 1.2 α 1C was checked, Na-K ATPase was detected as a membrane loading control. ( B ) Relative expression level of Rbfox2 was normalized with β-actin in differentially transfected cells, and presented as a bar chart. n = 4 independent experiments. ** P < 0.001, one-way ANOVA followed by a Tukey’s post hoc test. ( C ) Relative Ca V 1.2 α 1C membrane expression was normalized with Na-K ATPase in differentially-transfected cells. n = 4 independent experiments. P = 0.6679, one-way ANOVA followed by a Tukey’s post hoc test. ( D ) Raw traces of Ca V 1.2 whole-cell calcium current recorded from NRVMs treated with NT or Rbfox2 siRNA in 10 mmol/L Ba 2+ external solution. ( E ) I-V relationship of calcium channel current recorded under the different testing potential, increased from − 50 to 50 mV in NRVMs transfected with NT or Rbfox2 siRNA. ( F ) Ca V 1.2 channel current density in NRVMs was analyzed after transfected with NT or Rbfox2 siRNA. ( H ) Plots of steady-state activation (SSA) curve of Ca V 1.2 channel were analyzed from I-V currents in NT or Rbfox2 siRNA-treated NRVMs. ( H ) Plots of the steady-state inactivation (SSI) was also recorded in NRVMs. ( I ) Ca V 1.2 window currents were superimposed from SSI ( f ∞ ) and SSA ( d ∞ ) curves of NRVMs

Article Snippet: Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca V 1.2 α 1C (1.6 μg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 μg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus).

Techniques: Knockdown, Expressing, Isolation, Western Blot, Membrane, Control, Transfection, Activation Assay

Journal: Molecular Psychiatry

Article Title: Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits

doi: 10.1038/mp.2015.194

Figure Lengend Snippet: Dorsal striatum and dopamine homeostasis in the tgDISC1 rat. ( a ) Neurochemical quantification of post mortem dopamine (DA) in the dStr of tgDISC1 and negative control animals. TgDISC1 rats ( n =12) had lower levels of DA as compared with negative controls (NCs) ( n =12). TgDISC1 and negative control rats had 13 337±375 and 11 675±504 pg/mg DA. Unpaired t -test ** P =0.005. ( b ) Total D2 receptor abundance in the dStr. Autoradiography was performed with the D2R specific radioligand [ 3 H]raclopride. No difference in ligand binding and therefore total receptor density could be found in tgDISC1 rats ( n =10) and controls ( n =10) in the dorsal striatum. Mean receptor density was 870±26 fmol/mg protein for controls and 815±26 for tgDISC1 rats. Unpaired t -test P =0.156. ( c ) Elevated striatal D2High receptor portion in tgDISC1 rats. TgDISC1 rats ( n =6) had an 81% increase in D2High receptor portions compared with negative controls ( n =6) as measured by [ 3 H]domperidone binding. Binding of the radioligand was challenged with either 1 n M or 100 n M DA, concentrations at which no significant occupation of D2Rs or D2High-specific binding occurs, respectively. Proportions of D2High receptors in relation to total D2 receptors were 33±3.9% in NCs and 59.7±6.5% in TGs. Unpaired t -test ** P =0.005. ( d ) Peak DA release in the dStr of tgDISC1 rats. Fast-scan cyclic voltammetry measurement of DA in striatal slices revealed no difference in the peak release of DA in tgDISC1 rats (2.16±0.43 μ m ; n =4) and controls (1.88±0.58 μ m ; n =6). Mann–Whitney U -test P =0.762. ( e ) Clearance of extracellular DA in the dStr of tgDISC1 rats. TgDISC1 rats (tau=0.131±0.006; n =4) show increased extracellular DA clearance compared with negative controls (0.176±0.006; n =6) as measured by fast-scan cyclic voltammetry. Mann–Whitney U -test * P =0.036. ( f ) Striatal DAT levels in tgDISC1 rats. Preparation of the synaptic plasma membrane (SPM) and subsequent western blotting revealed a 19% increase in dopamine transporter levels in the dorsal striatum of tgDISC1 rats (NC, TG n =6). Densitometric analysis was performed by normalization of DAT to the Na/K-ATPase signal in the preparations. Unpaired t -test ** P =0.004. All means±s.e.m.

Article Snippet: For western blot: actin (rabbit, 1:10 000; Sigma-Aldrich); DAT (rat MAB369, 1:1 000; Merck Millipore); huDISC1 (mouse 14F2, 1:1 000; Korth lab ); ratDISC1 (rabbit hu-precleared C-term,1:1 000; Korth Lab ); Na/K-ATPase (rabbit, 1:1500; Cell Signaling; the antibody detects the α1 subunit.

Techniques: Negative Control, Autoradiography, Ligand Binding Assay, Binding Assay, MANN-WHITNEY, Clinical Proteomics, Membrane, Western Blot

Journal: Gut Microbes

Article Title: Hypoxia increases susceptibility of human intestinal epithelial cells to rotavirus infection through repression of interferon induction

doi: 10.1080/19490976.2025.2560593

Figure Lengend Snippet: Hypoxia induces a decreased TBK1 and IRF3 activation following rotavirus infection. T84 WT cells were cultured for 24 hours in normoxia (20% O 2 ) or hypoxia (1% O 2 ) and infected with rotavirus expressing UnaG at MOI 0.5 as determined in MA104 cells. Cells were harvested at indicated timepoints post-infection with RIPA buffer and total TBK1, phospho-TBK1 (pTBK1), total IRF3, phospho-IRF3 (pIRF3), and phospho-paxillin (pPAX) levels were assessed by immunoblotting. Ratio of phosphorylated protein to total protein indicated on blots. Actin used as a loading control. n ≥ 3 replicates. Representative image shown. N = normoxia, H = hypoxia.

Article Snippet: Primary antibodies include pTBK1 (Cell Signaling, 5483S, 1:1000), TBK1/NAK (Cell Signaling, 3013S, 1:1000), pIRF3 (Cell Signaling, 4947, 1:1000), IRF3 (Cell Signaling, 11904T, 1:1000), PP2ACa (Cell Signaling, 2038S, 1:2000), PPP2R5B (Santa Cruz 515,676, 1:2000), pPAX (Cell Signaling, 2541, 1:1000), STAT1 (BD 610,115, 1:1000), pSTAT1 (BD 612,233, 1:1000), β-actin (Sigma, A5441, 1:2000), α-tubulin (Sigma, T9026, 1:1000), LaminB1 (Santa Cruz 374,015, 1:200).

Techniques: Activation Assay, Infection, Cell Culture, Expressing, Western Blot, Control

Journal: Gut Microbes

Article Title: Hypoxia increases susceptibility of human intestinal epithelial cells to rotavirus infection through repression of interferon induction

doi: 10.1080/19490976.2025.2560593

Figure Lengend Snippet: PPP2R5B is essential for inhibition of IFN response in hypoxia. (A) Expression of PP2A subunits was measured by qRT-PCR in WT T84 cells after 48 hours in normoxia (20% O 2 ) or hypoxia (1% O 2 ). Expression in hypoxia plotted relative to normoxia. (B) Representative western blot of PPP2R5B protein levels in WT or PPP2R5B KO T84 cells following 48 hours of incubation in normoxia (20% O 2 ) or hypoxia (1% O 2 ), actin is used as a loading control and the quantification of protein relative to actin is shown below. (C) T84 WT and PPP2R5B KO cells were seeded for 24 hours in normoxia (20% O 2 ) or hypoxia (1% O 2 ) and infected with rotavirus expressing UnaG at MOI 0.25 as determined in MA104 cells. RNA was harvested at 8 hpi and qRT-PCR was performed to measure expression of IFNλ2/3. Relative expression was normalized to HPRT1. (D) T84 WT and PPP2R5B KO cells were seeded for 24 hours in normoxia (20% O 2 ) or hypoxia (1% O 2 ) prior to infection with rotavirus. Cells were harvested at indicated timepoints post-infection and total TBK1 and phopspho-TBK1 (pTBK1) levels were assessed by immunoblotting. Ratio of pTBK1/TBK1 indicated on blot. Actin is used as a loading control, representative blot shown. (E) WT T84 and PPP2R5B knockout T84 cells were seeded in normoxia (20% O 2 ) or hypoxia (1% O 2 ) and infected with MRV. RNA was harvested at 16 hpi and qRT-PCR was performed to measure expression of IFNλ2/3. Relative expression was normalized to HPRT1. (F) Representative western blot of PPP2R5B expression in WT, PPP2R5B KO, and PPP2R5B KO + PPP2R5B rescue cell lines, actin is used as a loading control and the quantification of protein relative to actin is shown below (G) WT T84, PPP2R5B KO, and PPP2R5B KO + PPP2R5B cells were seeded for 24 hours in normoxia (20% O 2 ) or hypoxia (1% O 2 ) and infected with rotavirus. RNA was harvested at 8 hpi and qRT-PCR was performed to measure expression of IFNλ2/3. Relative expression was normalized to HPRT1. (A-G) n ≥ 3 replicates. Significance determined by unpaired t test (normal distribution, in black) or mann-whitney (non-normal distribution, in blue) between normoxic and hypoxic conditions, ns = not significant, significant p values shown. N = normoxia; H = hypoxia.

Article Snippet: Primary antibodies include pTBK1 (Cell Signaling, 5483S, 1:1000), TBK1/NAK (Cell Signaling, 3013S, 1:1000), pIRF3 (Cell Signaling, 4947, 1:1000), IRF3 (Cell Signaling, 11904T, 1:1000), PP2ACa (Cell Signaling, 2038S, 1:2000), PPP2R5B (Santa Cruz 515,676, 1:2000), pPAX (Cell Signaling, 2541, 1:1000), STAT1 (BD 610,115, 1:1000), pSTAT1 (BD 612,233, 1:1000), β-actin (Sigma, A5441, 1:2000), α-tubulin (Sigma, T9026, 1:1000), LaminB1 (Santa Cruz 374,015, 1:200).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Incubation, Control, Infection, Knock-Out, MANN-WHITNEY

Journal: Microbiology Spectrum

Article Title: Gallic acid inhibits EMCV infection via targeting the interaction between TBK1 and IRF3 to promote IFN-β expression

doi: 10.1128/spectrum.01524-25

Figure Lengend Snippet: GA regulates the RLR signaling pathway to inhibit EMCV replication. ( A ) Compared with the cells treated with 0 µM GA, those treated with GA (80/160 µM) significantly upregulated the expression of TBK1, p-TBK1, IRF3, and p-IRF3. ( B–D ) The overexpression of TBK1 and IRF3 significantly increased the expression of IFN-β. GAPDH was used as a control. Data were presented as the mean ± standard deviation (SD) of three independent experiments with P values indicating the results of one-way ANOVA and Tukey’s multiple comparison test or two-tailed t tests. *** P < 0.001, ** P < 0.01, and * P < 0.05 vs EMCV group; ### P < 0.001, ## P < 0.01, and # P < 0.05 vs mock group.

Article Snippet: TBK1 antibody (3013S) and phospho-IRF3 (Ser386) (37829 S) were bought from Cell Signaling Technology.

Techniques: Expressing, Over Expression, Control, Standard Deviation, Comparison, Two Tailed Test

Journal: Microbiology Spectrum

Article Title: Gallic acid inhibits EMCV infection via targeting the interaction between TBK1 and IRF3 to promote IFN-β expression

doi: 10.1128/spectrum.01524-25

Figure Lengend Snippet: GA induces the expression of IFN-β by targeting IRF3 to promote the interaction between TBK1 and IRF3. ( A ) GA (160 µM) significantly upregulated the expressions of TBK1, p-TBK1, IRF3, and p-IRF3. ( B ) GA (160 µM) significantly upregulated the expression of IFN-β. ( C ) GA could promote the interaction between TBK1 and IRF3. ( D ) siRNA targeting IRF3 was determined by Western blotting. After siRNA targeting IRF3, GA had no significant effect on the virus titer ( E ) and the expression of IFN-β ( F ) during EMCV infection. Data were presented as the mean ± standard deviation (SD) of three independent experiments, and P values indicate the results of one-way ANOVA with Tukey’s multiple comparison test or two-tailed t tests. *** P < 0.001, ** P < 0.01, and * P < 0.05 vs EMCV group; ### P < 0.001, ## P < 0.01, and # P < 0.05 vs mock group.

Article Snippet: TBK1 antibody (3013S) and phospho-IRF3 (Ser386) (37829 S) were bought from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Virus, Infection, Standard Deviation, Comparison, Two Tailed Test

Journal: Microbiology Spectrum

Article Title: Gallic acid inhibits EMCV infection via targeting the interaction between TBK1 and IRF3 to promote IFN-β expression

doi: 10.1128/spectrum.01524-25

Figure Lengend Snippet: Schematic diagram showing that GA targets TBK1/IRF3 in vitro , promotes the expression of IFN-β, inhibits EMCV, and exhibits a protective effect on mice in vivo .

Article Snippet: TBK1 antibody (3013S) and phospho-IRF3 (Ser386) (37829 S) were bought from Cell Signaling Technology.

Techniques: In Vitro, Expressing, In Vivo