Journal: PLoS ONE

Article Title: Loss of caveolin-1 alters extracellular matrix protein expression and ductal architecture in murine mammary glands

doi: 10.1371/journal.pone.0172067

Figure Lengend Snippet: (A) Immunohistochemistry of fibronectin expression in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results demonstrated an increased abundance fibronectin fibers surrounding the ducts and in the adipose stroma of cav-1 -/- glands. Fibronectin expression was also localized to the epithelial cells (red arrows) of the ducts in both cav-1 +/+ and cav-1 -/- glands. (B) The percent expression of fibronectin was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. (C) The intensity of fibronectin staining was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. Scale bars are 50 μM and 20 μM in low and high magnification images, respectively. *p≤0.05. Fn: fibronectin. Results are reported as the mean ± the SEM.

Article Snippet: Antibodies used include: 1:150 anti-fibronectin (Abcam); 1:50 anti-tenascin-C (Abcam); 1:100 anti-αSMA (Abcam); and 1:25 CD31 (Abcam).

Techniques: Immunohistochemistry, Expressing, Staining

Journal: PLoS ONE

Article Title: Loss of caveolin-1 alters extracellular matrix protein expression and ductal architecture in murine mammary glands

doi: 10.1371/journal.pone.0172067

Figure Lengend Snippet: (A) The number of ducts were enumerated in αSMA stained sections from cav-1 -/- and cav-1 +/+ glands. Results demonstrated a highly significant average number of ducts per image in cav-1 -/- glands in comparison to cav-1 +/+ glands. (B) Images of fibronectin stained sections illustrate the abundance of large, elongated, branched ducts (red arrows) in cav-1 -/- glands. Ducts in cav-1 +/+ glands appear small and relatively round (red arrows). Scale bars are 100μM. (C) H&E stained images of glands demonstrate the presence of stacked ductal epithelial cells in cav-1 -/- sections, indicative of ductal hyperplasia. Cav-1 +/+ glands exhibit a continuous monolayer of ductal epithelial cells. Scale bars are 20μM. (D) The ductal circumference, measured as the region immediately outside the myoepithelial compartment, was analyzed in αSMA stained sections in cav-1 -/- and cav-1 +/+ glands. The ductal circumference was significantly greater in cav-1 -/- as opposed to cav-1 +/+ glands. (E) The area, measured as the region between the myoepithelial and luminal compartment, was analyzed in αSMA stained sections in cav-1 -/- and cav-1 +/+ glands. The ductal area was significantly greater in cav-1 -/- as opposed to cav-1 +/+ glands. (F) This image of an αSMA stained cav-1 +/+ duct illustrates the vectors used to assign stromal and lumen outlines used to analyze ductal measurements. The red line shows the stromal border while the blue line shows the lumen border. Scale bar is 50uM. *p≤0.05, **p≤0.01, ***p≤0.001. Results are reported as the mean ± the SEM.

Article Snippet: Antibodies used include: 1:150 anti-fibronectin (Abcam); 1:50 anti-tenascin-C (Abcam); 1:100 anti-αSMA (Abcam); and 1:25 CD31 (Abcam).

Techniques: Staining

Journal: Targeted Oncology

Article Title: Recombinant TIMP-1-GPI inhibits growth of fibrosarcoma and enhances tumor sensitivity to doxorubicin

doi: 10.1007/s11523-013-0294-5

Figure Lengend Snippet: Effect of locally applied TIMP-1-GPI on tumor proliferation and angiogenesis in vivo. a Exemplary pictures of anti-CD31 and anti-Ki67 antibodies staining. CD31 ( black arrowheads ) and Ki67 positive signals are less in TIMP-1-GPI treated group; b Ki67 index decreased significantly after TIMP-1-GPI treatment compared with vehicle control and rhTIMP-1 groups (vehicle control vs. TIMP-1-GPI, * p = 0.01; rhTIMP-1 vs. TIMP-1-GPI, ** p = 0.002)

Article Snippet: The antibodies used included monoclonal rabbit anti-Ki67 antibody (ab16667, Abcam, USA), polyclonal rabbit anti-CD31 antibody (ab28364, Abcam, USA).

Techniques: In Vivo, Staining

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Spinal cord astrocyte identification and high mobility group box-1 (HMGB1) knockdown. a Spinal cord astrocytes were identified using immunofluorescence. The percentage of cells stained with the astrocytic marker S100β, which were identified as astrocytes, was more than 95% of the total cells (three replicates). b HMGB1 knockdown efficiency in the plasma membrane and cytoplasm of spinal cord astrocytes was evaluated using Western blot for HMGB1 protein levels. Results were obtained after 72 h of specific HMGB1 shRNA treatment. HMGB1 protein levels were decreased to approximately 30% of normal levels with shRNA multiplicity of infection 60 as compared to normal astrocytes. * P < 0.05 vs. normal group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Immunofluorescence, Staining, Marker, Western Blot, shRNA, Infection

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of oxygen-glucose deprivation/reoxygenation (OGD/R) on cellular swelling, high mobility group box-1 (HMGB1), and aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes as well as levels of HMGB1 and interleukin-6 (IL-6) released into the surrounding medium. a Astrocyte volume measurement was performed using a Live Cell Imaging System. Cellular volume was calculated by the average value of four measured diameters of the largest compiled Z-slice image. Cellular volumes of spinal cord astrocytes were significantly increased at 2, 6, 12, 24, and 48 h during reoxygenation after OGD when compared with normal astrocytes. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). b Membrane and cytoplasmic HMGB1 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). c Membrane and cytoplasmic AQP4 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). d HMGB1 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates). e IL-6 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. # P < 0.05 vs. normal group; * P < 0.05 vs. OGD6h/R24h group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, Live Cell Imaging

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on cellular swelling in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R). a Astrocyte volume analysis was performed using a Live Cell Imaging System, and cellular volume was calculated by the average value of four measured diameters. Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly blocked increases in cellular volume of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation when compared with astrocytes of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b — d Effects of inhibiting HMGB1 on spinal cord astrocytic morphology and ultrastructure were evaluated using transmission electron microscopy at 6, 12, and 24 h during reoxygenation after OGD. After OGD/R, spinal cord astrocytes showed swelling at 6, 12, and 24 h during reoxygenation. The mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and an increase in the number of lysosomes (L) were concurrent with this observation. However, astrocytic swelling, mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and the increase in lysosome (L) number after OGD/R were reduced by HMGB1 inhibition using either HMGB1 shRNA or EP (× 50,000, bar equal to 1 μm, three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Cell Culture, Live Cell Imaging, shRNA, Transmission Assay, Electron Microscopy, Inhibition

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of inhibiting high mobility group box-1 (HMGB1) on HMGB1, aquaporin-4 (AQP4), and toll-like receptor-4 (TLR4) expression in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R) as well as levels of HMGB1 and interleukin-6 (IL-6) release into the surrounding medium. a Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly suppressed the increased levels of HMGB1 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 significantly suppressed the increased levels of AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 significantly suppressed increased levels of TLR4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates). d HMGB1, AQP4, and TLR4 immunofluorescence on spinal cord astrocytes at 24 h into the reoxygenation process after OGD showed significantly increased membrane and cytoplasmic levels of HMGB1, AQP4, and TLR4 in the OGD/R group when compared with those in the normal group. These were markedly suppressed in both the OGD/R + HMGB1 shRNA and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e , f Inhibiting HMGB1 mitigated increases in levels of HMGB1 and IL-6 in the surrounding medium when compared with levels in the OGD/R group at 6, 12, and 24 h during reoxygenation after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of either inhibiting high mobility group box-1 (HMGB1) or toll-like receptor-4 (TLR4) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, TLR4, myeloid differentiation primary response gene 88 (MyD88), aquaporin-4 (AQP4) upregulation, and nuclear factor-kappa B (NF-κB) activation as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a Inhibiting HMGB1 (using either HMGB1 shRNA or ethyl pyruvate (EP)) or TLR4 (using CLI-095 or C34) significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those in the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 or TLR4 significantly suppressed the increased levels of TLR4, MyD88, and AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 24 h during the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 or TLR4 significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). d Immunofluorescence results showed that either inhibiting HMGB1 or TLR4 decreased membrane and cytoplasmic TLR4 and AQP4 upregulation and attenuated the increases of nuclear NF-κB when compared with the OGD/R group at 24 h during reoxygenation (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). e Inhibiting HMGB1 or TLR4 reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Activation Assay, shRNA, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of nuclear factor-kappa B (NF-κB) inhibition on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, NF-κB activation, and aquaporin-4 (AQP4) upregulation, as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a NF-κB inhibition (using BAY 11-7082) significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation phase after OGD. * P < 0.05 vs. OGD/R group (three replicates). b NF-κB and AQP4 immunofluorescence in spinal cord astrocytes after 24 h of the reoxygenation process after OGD showed significantly increased nuclear levels of NF-κB and membrane and cytoplasmic levels of AQP4 in the OGD/R group. Levels were markedly attenuated in the OGD/R + HMGB1 shRNA, OGD/R + BAY 11-7082, and OGD/R + EP groups (× 200, bar equal to 100 μm). * P < 0.05 vs. OGD/R group (three replicates). c NF-κB inhibition significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those of the OGD/R group. * P < 0.05 vs. OGD/R group (three replicates). d NF-κB inhibition significantly suppressed increased AQP4 levels in both the plasma membrane and cytoplasm of spinal cord astrocytes after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates). e NF-κB inhibition reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. * P < 0.05 vs. OGD/R group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Inhibition, Activation Assay, Immunofluorescence, shRNA

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of recombinant HMGB1 (rHMGB1) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. Incubation of cultured spinal cord astrocytes with rHMGB1 (0, 0.1, 1, 10, and 20 ng/ml) for 24 h did not induce dose-dependent increases in the membrane and cytoplasmic AQP4 expression (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Recombinant, Expressing, Cell Culture, Incubation

Journal: Journal of Neuroinflammation

Article Title: Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

doi: 10.1186/s12974-017-1008-1

Figure Lengend Snippet: Effects of interleukin-6 (IL-6) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. a Spinal cord astrocytes were exposed to exogenous IL-6 at 0, 0.1, 1, or 10 ng/ml. After 24 h exposure, the membrane and cytoplasmic AQP4 expression in spinal cord astrocytes were markedly increased in the IL-6 0.1 ng/ml group, IL-6 1 ng/ml group, and IL-6 10 ng/ml group. * P < 0.05 vs. 0 ng/ml group (three replicates). b IL-6 levels increased in the surrounding medium of the OGD/R group after 24 h of the reoxygenation process after OGD. In comparison, this increase was significantly reduced in the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. OGD/R group (three replicates). c The effects of astrocyte conditioned medium (ACM) on AQP4 expression in cultured spinal cord astrocytes. Twenty-four hours exposure of spinal cord astrocytes to the ACM obtained from the OGD/R group significantly increased the membrane and cytoplasmic AQP4 expression when compared with astrocytes incubated with the ACM obtained from the OGD/R + HMGB1 shRNA group. * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates). d Western blot analysis showed that the neutralizing anti-rat-IL-6 antibody could significantly reverse the upregulation effect of exogenous IL-6 or OGD/R ACM containing increased IL-6 on AQP4 expression in cultured spinal cord astrocytes. # P < 0.05 vs. astrocytes + IL-6 group; * P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates)

Article Snippet: Primary antibodies include rabbit polyclonal anti-HMGB1 antibody for rat, mouse, and human (Abcam, Cat# ab18256, RRID:AB_444360, Cambridge, UK); rabbit polyclonal anti-AQP4 antibody for rat, mouse, human, and pig (Abcam, Cat# ab46182, RRID: AB_955676); mouse monoclonal anti-TLR4 antibody for rat, mouse, human, pig, baboon, bovine, and Chinese hamster (Novus, Cat# 76B357.1, RRID: AB_839000, Littleton, CO, USA); rabbit polyclonal anti-TLR4 antibody for rat, mouse, human, and rabbit (Boster, Cat# BA1717, RRID:AB_2716293); rabbit polyclonal anti-myeloid differentiation primary response gene 88 (MyD88) antibody for rat and human (Abcam, Cat# ab131071, RRID: AB_11156885); mouse monoclonal anti-IκBα antibody for rat, mouse, human, monkey, bovine, pig, and guinea pig (Cell Signaling Technology, Cat# 4814, RRID: AB_390781, Boston, MA, USA); mouse monoclonal anti-p-IκBα antibody for rat, mouse, human, and monkey (Cell Signaling Technology, Cat# 9246, RRID:AB_2267145); rabbit monoclonal anti-NF-κB antibody for rat, mouse, human, monkey, and bovine (Cell Signaling Technology, Cat# 4764, RRID:AB_823578); mouse monoclonal anti-GAPDH antibody for rat, mouse, and human (Beyotime, Cat# AF0006, RRID: AB_2715590, Shanghai, China); mouse monoclonal anti-Histone H3 antibody for rat, mouse, and human (Beyotime, Cat# AF0009, RRID: AB_2715593); and mouse monoclonal anti-S100β antibody for rat, mouse, human, rabbit, and pig (Boster, Cat# BM0120, RRID:AB_2716291).

Techniques: Expressing, Cell Culture, shRNA, Incubation, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: OPTN expression is downregulated in AD patients and APP/PS1 transgenic mice. A – D Transcriptome data of the entorhinal cortex, hippocampus, frontal cortex, and temporal cortex in AD patients were analyzed after normalization. E – H Nine-month-old APP/PS1 transgenic mice were anesthetized and euthanized to obtain the cerebral cortex and hippocampus. E Expression of OPTN in the cerebral cortex and hippocampus of APP/PS1 transgenic mice was detected by qRT-PCR using GAPDH as an internal control. F The protein level of OPTN in the cerebral cortex and hippocampus of APP/PS1 transgenic mice was assessed by western blotting using β-actin as an internal control. G ImageJ software was used to semiquantitatively analyze the fold change of OPTN relative to β-actin. H WT or APP/PS1 Tg mice were double-stained for Iba1 (green) and OPTN (red). I Expression of OPTN in microglia in the cortex and hippocampus of APP/PS1 transgenic mice at 9 months of age was detected by flow cytometry. The data represent the means ± S.E. of independent experiments. APP/PS1 transgenic mice were compared with WT mice * P < 0.05, ** P < 0.01

Article Snippet: Other antibodies, including β-actin (#3700, mouse, 1:2,000 for WB), Histone (#4499, rabbit, 1:2000 for WB), IL-1β (#12242, mouse, 1:2000 for WB), RIPK1 (#3493, rabbit, 1:2000 for WB), p-IKBα (#2859, rabbit, 1:2000 for WB), IKBα (#4814, mouse, 1:2000 for WB), GFAP (#80788, rabbit, 1:5000 for WB), ubiquitin (#3933, rabbit, 1:1000 for WB) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR, Control, Western Blot, Software, Staining, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: OPTN alleviates Aβo-induced dysfunction of mitochondrial autophagy. A , B BV2 cells were treated with Aβo in the absence or presence of transfection with plvx-IRES-OPTN. A mKeima fluorescence was evoked using two excitation filters (438 ± 12 nm and 550 ± 15 nm) and a 610LP emission filter. B Expression levels of HSP60 and OPTN were detected by western blotting. β-actin served as an internal control. The data represent the means ± S.E. of independent experiments. BV2 cells treated with vehicle group or Aβo and overexpressing OPTN were compared with group Aβo alone * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Other antibodies, including β-actin (#3700, mouse, 1:2,000 for WB), Histone (#4499, rabbit, 1:2000 for WB), IL-1β (#12242, mouse, 1:2000 for WB), RIPK1 (#3493, rabbit, 1:2000 for WB), p-IKBα (#2859, rabbit, 1:2000 for WB), IKBα (#4814, mouse, 1:2000 for WB), GFAP (#80788, rabbit, 1:5000 for WB), ubiquitin (#3933, rabbit, 1:1000 for WB) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Fluorescence, Expressing, Western Blot, Control

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: The AIM2 inflammasome is activated in AD patients and APP/PS1 transgenic mice. A – D Brain transcriptome data from patients with AD and controls were collected from the GEO database and normalized for analysis. E – H APP/PS1 transgenic mice at the age of 9 months were anesthetized and euthanized to obtain the cerebral cortex and hippocampus. E Detection of the expression levels of AIM2, ASC, pro-caspase-1, caspase-1 and IL-1β in the cerebral cortex by western blotting. β-actin served as the internal control. In the right panel, ImageJ software was used to semiquantitatively analyze the western blotting results. F qRT-PCR was used to detect the mRNA expression of AIM2 and ASC in the cerebral cortex. GAPDH served as internal control. G The expression of AIM2, ASC, pro-caspase-1, caspase-1 and IL-1β in the hippocampus was detected by western blotting. β-actin served as the internal control. H The mRNA expression of AIM2 and ASC in the hippocampus was detected by qRT-PCR. GAPDH was used as the internal control. The data present means ± S.E. of independent experiment. APP/PS1 transgenic mice were compared with WT mice * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Other antibodies, including β-actin (#3700, mouse, 1:2,000 for WB), Histone (#4499, rabbit, 1:2000 for WB), IL-1β (#12242, mouse, 1:2000 for WB), RIPK1 (#3493, rabbit, 1:2000 for WB), p-IKBα (#2859, rabbit, 1:2000 for WB), IKBα (#4814, mouse, 1:2000 for WB), GFAP (#80788, rabbit, 1:5000 for WB), ubiquitin (#3933, rabbit, 1:1000 for WB) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transgenic Assay, Expressing, Western Blot, Control, Software, Quantitative RT-PCR

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: Aβo activates the AIM2 inflammasome. A – D BV2 cells were treated with Aβo for 12 h. A Expression levels of AIM2, ASC, pro-caspase-1 and caspase-1 were detected by western blotting. β-actin served as an internal control. B ImageJ software was used for semiquantitative analysis of western blots. C qRT-PCR was used to detect the mRNA expression of AIM2 and ASC with GAPDH as an internal control. D Secretion of IL-1β was evaluated by ELISA. E – H Primary microglia were treated with Aβo for 12 h. E Protein levels of AIM2, ASC, pro-caspase-1, and caspase-1 were detected by western blotting with β-actin as the internal control. F ImageJ software was used to semiquantitatively analyze the fold change in AIM2, ASC, pro-caspase-1 and caspase-1 relative to β-actin. G mRNA expression of AIM2 and ASC was detected by qRT-PCR with GAPDH as an internal control. H Secretion of IL-1β was detected by ELISA. The data present means ± S.M. of independent experiment. Aβo treatment were compared with vehicle treatment * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Other antibodies, including β-actin (#3700, mouse, 1:2,000 for WB), Histone (#4499, rabbit, 1:2000 for WB), IL-1β (#12242, mouse, 1:2000 for WB), RIPK1 (#3493, rabbit, 1:2000 for WB), p-IKBα (#2859, rabbit, 1:2000 for WB), IKBα (#4814, mouse, 1:2000 for WB), GFAP (#80788, rabbit, 1:5000 for WB), ubiquitin (#3933, rabbit, 1:1000 for WB) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control, Software, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: OPTN alleviates the Aβo-induced AIM2 inflammasome. A – G OPTN was silenced in BV2 cells that were then treated with Aβo for 4, 8, and 12 h. A Western blotting was used to detect the protein expression of AIM2, ASC, OPTN and pro-caspase-1 in whole-cell lysates. Meanwhile, extracellular secretion of caspase-1 and IL-1β was assessed in the conditioned medium. B – G ImageJ software was used to semiquantitatively analyze the fold changes in caspase-1, IL-1β, AIM2, ASC, OPTN and pro-caspase-1 relative to β-actin. The data are presented as the mean ± S.M. of independent experiment. WT BV2 cells compared to OPTN knockdown, * P < 0.05, ** P < 0.01, *** P < 0.001. H – N BV2 cells gradually increased their expression of OPTN in the absence or presence of Aβo for 12 h. H Western blotting was used to detect the protein expression of AIM2, ASC, OPTN and pro-caspase-1 in whole-cell lysates. Meanwhile, extracellular secretion of caspase-1 and IL-1β was detected in the cell culture medium. I – N ImageJ software was used to semiquantitatively analyze the fold change in caspase-1, IL-1β, AIM2, ASC, OPTN and pro-caspase-1 relative to β-actin. The data are presented as the means ± S.M. of independent experiment. The data present mean ± S.M. of independent experiment. Aβo treatment BV2 cells compared with vehicle treatment BV2 cells, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Other antibodies, including β-actin (#3700, mouse, 1:2,000 for WB), Histone (#4499, rabbit, 1:2000 for WB), IL-1β (#12242, mouse, 1:2000 for WB), RIPK1 (#3493, rabbit, 1:2000 for WB), p-IKBα (#2859, rabbit, 1:2000 for WB), IKBα (#4814, mouse, 1:2000 for WB), GFAP (#80788, rabbit, 1:5000 for WB), ubiquitin (#3933, rabbit, 1:1000 for WB) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Software, Knockdown, Cell Culture

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: The Toll-like receptor pathway is upregulated in AD patients, and RIPK1 expression is increased in APP/PS1 transgenic mice. A – D Transcriptome sequencing data of the entorhinal cortex, hippocampal cortex, temporal cortex and frontal cortex tissues from AD patients were collected from the GEO database, and GSEA was performed after normalization. E – H The differences in RIPK1 expression in the transcriptome of the entorhinal cortex, hippocampus, frontal cortex and temporal cortex were normalized in AD patients. I – K Nine-month-old APP/PS1 Tg mice were anesthetized and euthanized to obtain the cerebral cortex and hippocampus. I mRNA expression of RIPK1 in the cerebral cortex and hippocampus was detected by qPCR using GAPDH as the internal control. J Protein levels of RIPK1 in the cerebral cortex and hippocampus were elucidated by western blotting with β-actin as the internal control. k ImageJ software was used to semiquantitatively analyze the fold change in RIPK1 relative to β-actin. The data present means ± S.M. of independent experiment. The data present mean ± S.M. of independent experiment. APP/PS1 Tg mice compared with WT mice, *** P < 0.001

Article Snippet: Other antibodies, including β-actin (#3700, mouse, 1:2,000 for WB), Histone (#4499, rabbit, 1:2000 for WB), IL-1β (#12242, mouse, 1:2000 for WB), RIPK1 (#3493, rabbit, 1:2000 for WB), p-IKBα (#2859, rabbit, 1:2000 for WB), IKBα (#4814, mouse, 1:2000 for WB), GFAP (#80788, rabbit, 1:5000 for WB), ubiquitin (#3933, rabbit, 1:1000 for WB) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Transgenic Assay, Sequencing, Control, Western Blot, Software

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: OPTN negatively regulates RIPK1 inflammatory signaling pathways. A BV2 cells with OPTN silenced were treated with Aβo for 12 h. Then, OPTN, RIPK1, p-IκBα, and IκBα in the cytoplasm and NF-κB in the cytoplasm or nucleus were detected by western blot with β-actin as an internal control. B – G ImageJ software was used to semiquantitatively analyze the optical density of western blots. H – K OPTN-silenced BV2 cells were treated with Aβo for 12 h. H OPTN mRNA expression was detected by qRT-PCR using GAPDH as an internal control. I RIPK1 RNA expression was detected by qRT-PCR using GAPDH as an internal control. J Extracellular secretion of IL-1β was assessed by ELISA. K The binding activity of NF-κB was evaluated by dual-luciferase assay. The data are presented as the means ± S.M. of independent experiment. OPTN-silenced BV2 cells compared to control BV2 cells or Aβo-treated BV2 cells compared to vehicle BV2 cells, *P < 0.05, ** P < 0.01, *** P < 0.001. L – V BV2 cells with ectopic overexpression of OPTN in the absence or presence of Aβo treatment for 12 h. L Protein levels of OPTN, RIPK1, p-IκBα, and IκBα in the cytoplasm and NF-κB in the cytoplasm or nucleus were detected by western blot using β-actin as an internal control. N – R ImageJ software was used to semiquantitatively analyze the western blot results. S mRNA expression of OPTN was detected by qRT-PCR using GAPDH as an internal control. T mRNA expression of RIPK1 was detected by qRT-PCR using GAPDH as an internal control. U Extracellular secretion of IL-1β was assessed by ELISA. V The binding activity of NF-κB was evaluated using a dual-luciferase assay. The data present means ± S.M. of independent experiment. OPTN overexpressed BV2 cells compared with control BV2 cells or Aβo-treated BV2 cells compared with vehicle BV2 cells, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Other antibodies, including β-actin (#3700, mouse, 1:2,000 for WB), Histone (#4499, rabbit, 1:2000 for WB), IL-1β (#12242, mouse, 1:2000 for WB), RIPK1 (#3493, rabbit, 1:2000 for WB), p-IKBα (#2859, rabbit, 1:2000 for WB), IKBα (#4814, mouse, 1:2000 for WB), GFAP (#80788, rabbit, 1:5000 for WB), ubiquitin (#3933, rabbit, 1:1000 for WB) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Protein-Protein interactions, Western Blot, Control, Software, Expressing, Quantitative RT-PCR, RNA Expression, Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Luciferase, Over Expression

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: OPTN degrades RIPK1 through the ubiquitin proteasome pathway. A BV2 cells overexpressing OPTN were treated with bafilomycin A1 (200 nM) or MG132 (10 μM) for 6 h. The expression of RIPK1 and OPTN was detected by western blotting using β-actin as an internal control. B ImageJ software was used to semiquantitatively analyze the western blot results. C Flag-RIPk1 was ectopically expressed in OPTN-silenced BV2 cells, which were then immunoprecipitated using an anti-Flag antibody derived from mice. The precipitated protein was further detected using a ubiquitin antibody derived from rabbits. Flag antibody was used to detect RIPK1, and OPTN was used to detect the OPTN protein levels in the total protein. In the right panel, ImageJ software was used to semiquantitatively analyze ubiquitin levels. D OPTN was ectopically overexpressed in Flag-RIPk1-overexpressing BV2 cells, which were then immunoprecipitated using an anti-Flag antibody derived from mice. The precipitated protein was detected using rabbit-derived ubiquitin antibody. Flag antibodies were used to detect the protein levels of RIPK1, and OPTN was used to detect OPTN levels in whole-cell lysates. In the right panel, ImageJ software was used to semiquantitatively analyze ubiquitin levels. The data present means ± S.M. of independent experiment. OPTN silenced or overexpressed BV2 cells compared with control group, * P < 0.05, ** P < 0.01

Article Snippet: Other antibodies, including β-actin (#3700, mouse, 1:2,000 for WB), Histone (#4499, rabbit, 1:2000 for WB), IL-1β (#12242, mouse, 1:2000 for WB), RIPK1 (#3493, rabbit, 1:2000 for WB), p-IKBα (#2859, rabbit, 1:2000 for WB), IKBα (#4814, mouse, 1:2000 for WB), GFAP (#80788, rabbit, 1:5000 for WB), ubiquitin (#3933, rabbit, 1:1000 for WB) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Control, Software, Immunoprecipitation, Derivative Assay

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: Overexpression of OPTN in the brains of APP/PS1 transgenic mice alleviates activation of the AIM2 inflammasome and RIPK1 pathways. A – D Three-month-old APP/PS1 transgenic mice were injected with OPTN or control adeno-associated virus in the hippocampus and cortex for 1 month. Brain tissue was collected after anesthesia euthanasia. A , B Western blotting was used to detect the protein expression of OPTN, AIM2, ASC and caspase-1 in the cerebral cortex and hippocampus of mice. β-actin served as the internal control. C , D Western blot analysis was used to determine levels of RIPK1, GFAP, p-IKBα, IKBα, IL-1β, and NF-κB in the cytoplasm and nucleus of mice. Histone and β-actin were used as internal controls for the nucleus and cytoplasm, respectively. The data present means ± S.M. of independent experiment. OPTN-AAV injected APP/PS1 Tg mice compared with control-AAV injected APP/PS1 Tg mice, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Other antibodies, including β-actin (#3700, mouse, 1:2,000 for WB), Histone (#4499, rabbit, 1:2000 for WB), IL-1β (#12242, mouse, 1:2000 for WB), RIPK1 (#3493, rabbit, 1:2000 for WB), p-IKBα (#2859, rabbit, 1:2000 for WB), IKBα (#4814, mouse, 1:2000 for WB), GFAP (#80788, rabbit, 1:5000 for WB), ubiquitin (#3933, rabbit, 1:1000 for WB) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, Transgenic Assay, Activation Assay, Injection, Control, Virus, Western Blot, Expressing