rabbit anti nf κb p65  (Cell Signaling Technology Inc)

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Antibodies used for western blotting

Article Snippet: rabbit anti-NF-κB p65 , 1: 1000 , Cell Signaling Technology , 8242 , AB_10859369.

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Tryp promotes the M2 polarization of microglia through the cGAS/STING/NF-κB pathway. (A) Western blot analysis of cGAS and p-STING in BV2 cells incubated with Ctrl, LPS + DMSO (LD group) and LPS + Tryp (10.0 μM; LT group) for 1 hour. (B, C) Quantitative analysis of cGAS (B) and p-STING (C) protein levels as shown in A (normalized to β-actin, n = 8 individual samples of cells per group). (D) Western blot analysis of p-p65, p65, p-IκBα and IκBα in BV2 cells incubated with Ctrl, LD and LT for 1 hour. (E–H) Quantitative analysis of p-p65 (E, normalized to p65), p65 (F, normalized to β-actin), p-IκBα (G, normalized to IκBα), and IκBα (H, normalized to β-actin) protein levels shown in (D) (p-p65 and p65, n = 11 individual samples of cells per group; p-IκBα and IκBα, n = 8 individual samples of cells per group). (I) Western blot analysis of cGAS and p-STING in BV2 cells treated with Ctrl, LD, LT and LPS + Tryp + SR-717 (20.0 μM; LT + SR-717) for 1 hour. (J, K) Quantitative analysis of cGAS (J) and p-STING (K) protein levels shown in I (normalized to β-actin, n = 6 individual samples of cells per group). (L) Western blot analysis of p-p65, p65, p-IκBα and IκBα in BV2 cells treated with Ctrl, LD, LT and LT + SR-717 for 1 hour. (M–P) Quantitative analysis of p-p65 (M, normalized to p65), p65 (N, normalized to β-actin), p-IκBα (O, normalized to IκBα) and IκBα (P, normalized to β-actin) protein levels shown in L ( n = 6 individual samples of cells per group). (Q–U) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (Q), IL-1 β (R), TNF-α (S), CD206 (T) and Arg-1 (U) mRNA expression levels within BV2 cells treated with Ctrl, LD, LT and LT + SR-717 for 24 hours (normalized to β-actin, n = 3 individual samples of cells per group). (V) Immunostaining of CD86 (CD86 + BV2 cells and CD86 + primary cultured microglia were both marked by white arrows) and CD206 (CD206 + BV2 cells and CD206 + primary cultured microglia were both marked by white arrows) in BV2 cells and primary cultured microglia treated with Ctrl, LD, LT and LT + SR-717 for 24 hours. Scale bar: 100 μm. (W, X) Quantitative analysis of the percentages of CD86 + and CD206 + BV2 cells (W; n = 6 individual samples of cells per group), and primary cultured microglia (X; n = 3 individual samples of cells per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data were from at least three separate and independent studies. Arg-1: Arginase-1; CD206: cluster of differentiation 206; cGAS: cyclic GMP-AMP synthase; Ctrl: control; DMSO: dimethyl sulfoxide; IκBα: nuclear factor-κB inhibitory protein α; IL-6: interleukin-6; ns: not significant; LPS: lipopolysaccharide; p: phosphorylated; STING: stimulator of interferon genes; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

Article Snippet: rabbit anti-NF-κB p65 , 1: 1000 , Cell Signaling Technology , 8242 , AB_10859369.

Techniques: Western Blot, Incubation, Reverse Transcription, Polymerase Chain Reaction, Expressing, Immunostaining, Cell Culture, Control

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Tryp shifts microglia from M1 to M2 phenotype after spinal cord injury by targeting the cGAS/STING/NF-κB pathway. (A) Western blot analysis of cGAS and p-STING in spinal cord treated with Tryp at 3 dpi. (B) Quantitative analysis of cGAS and p-STING protein levels shown in A (normalized to β-actin, n = 6 mice per group). (C, E) Confocal laser scanning microscopy double immunostaining images (60× magnification) of cGAS (purple) and Iba1 (green) (C), and p-STING (purple) and Iba1 (green) (E) in the spinal cord lesion area of Sham + DMSO (T8–T10), Sham + Tryp (T8–T10), SCI + DMSO and SCI + Tryp mice at 3 dpi. Higher magnification panels are single optical Z sections of the areas marked by boxes. cGAS + Iba1 + cells and p-STING + Iba1 + cells were marked by white arrowheads. Scale bars, 50 μm (left) and 20 μm (enlarge). (D, F) Quantitative analysis of the percentages of cGAS + Iba1 + cells in Iba1 + cells (D, n = 3 mice per group), and p-STING + Iba1 + cells in Iba1 + cells (F, n = 3 mice per group). (G) Western blot analysis of p-p65, p65, p-IκBα and IκBα in spinal cord treated with Tryp at 7 dpi. (H) Quantitative analysis of p-p65 (normalized to p65), p65 (normalized to β-actin), p-IκBα (normalized to IκBα) and IκBα (normalized to β-actin) protein levels shown in G ( n = 10 mice per group). (I, K) Confocal laser scanning microscopy double immunostaining images (60× magnification) of CD86 (red) and CD68 (green) (I), and CD206 (red) and CD68 (green) (K) in the spinal cord lesion area of Sham + DMSO (T8–T10), Sham + Tryp (T8–T10), SCI + DMSO and SCI + Tryp mice at 7 dpi. Higher magnification panels are single optical Z sections of the areas marked by boxes. CD86 + CD68 + cells and CD206 + CD68 + cells were marked by white arrowheads. Scale bars: 50 μm (left) and 20μm (enlarge). (J, L) Quantitative analysis of the percentages of CD68 + /DAPI (left) and CD86 + CD68 + /CD68 + (right) (J, n = 6 mice per group), and CD68 + /DAPI (left) and CD206 + CD68 + /CD68 + (right) (L, n = 6 mice per group). Data were analyzed by one-way analysis of variance with Tukey’s multiple comparisons test (left) and unpaired two-tailed Student’s t -test (right). (M) Western blot analysis of CD206 and CD86 expression in spinal cord treated with Tryp at 7 dpi. (N, O) Quantitative analysis of CD206 (N) and CD86 (O) protein levels shown in M (normalized to β-actin, n = 9 mice per group). (P–T) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (P), IL-1 β (Q), TNF-α (R), CD206 (S) and Arg-1 (T) gene expression levels in spinal cords of Sham + DMSO, Sham + Tryp, SCI + DMSO and SCI + Tryp mice at 7 dpi (normalized to β-actin, n = 6 mice per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data were from at least three separate and independent studies. Arg-1: Arginase-1; CD206: cluster of differentiation 206; cGAS: cyclic GMP-AMP synthase; DMSO: dimethyl sulfoxide; dpi: days post-injury; Iba1: ionized calcium-binding adapter molecule 1; IL-6: interleukin-6; ns: not significant; NF-κB: nuclear factor-κB; ns: not significant; p: phosphorylated; p-STING: phosphorylated stimulator of interferon genes; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

Article Snippet: rabbit anti-NF-κB p65 , 1: 1000 , Cell Signaling Technology , 8242 , AB_10859369.

Techniques: Western Blot, Confocal Laser Scanning Microscopy, Double Immunostaining, Two Tailed Test, Expressing, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Binding Assay

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Antibodies used for western blotting

Article Snippet: rabbit anti-p-NF-κB p65 , 1: 1000 , Cell Signaling Technology , 3033 , AB_331284.

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Tryp promotes the M2 polarization of microglia through the cGAS/STING/NF-κB pathway. (A) Western blot analysis of cGAS and p-STING in BV2 cells incubated with Ctrl, LPS + DMSO (LD group) and LPS + Tryp (10.0 μM; LT group) for 1 hour. (B, C) Quantitative analysis of cGAS (B) and p-STING (C) protein levels as shown in A (normalized to β-actin, n = 8 individual samples of cells per group). (D) Western blot analysis of p-p65, p65, p-IκBα and IκBα in BV2 cells incubated with Ctrl, LD and LT for 1 hour. (E–H) Quantitative analysis of p-p65 (E, normalized to p65), p65 (F, normalized to β-actin), p-IκBα (G, normalized to IκBα), and IκBα (H, normalized to β-actin) protein levels shown in (D) (p-p65 and p65, n = 11 individual samples of cells per group; p-IκBα and IκBα, n = 8 individual samples of cells per group). (I) Western blot analysis of cGAS and p-STING in BV2 cells treated with Ctrl, LD, LT and LPS + Tryp + SR-717 (20.0 μM; LT + SR-717) for 1 hour. (J, K) Quantitative analysis of cGAS (J) and p-STING (K) protein levels shown in I (normalized to β-actin, n = 6 individual samples of cells per group). (L) Western blot analysis of p-p65, p65, p-IκBα and IκBα in BV2 cells treated with Ctrl, LD, LT and LT + SR-717 for 1 hour. (M–P) Quantitative analysis of p-p65 (M, normalized to p65), p65 (N, normalized to β-actin), p-IκBα (O, normalized to IκBα) and IκBα (P, normalized to β-actin) protein levels shown in L ( n = 6 individual samples of cells per group). (Q–U) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (Q), IL-1 β (R), TNF-α (S), CD206 (T) and Arg-1 (U) mRNA expression levels within BV2 cells treated with Ctrl, LD, LT and LT + SR-717 for 24 hours (normalized to β-actin, n = 3 individual samples of cells per group). (V) Immunostaining of CD86 (CD86 + BV2 cells and CD86 + primary cultured microglia were both marked by white arrows) and CD206 (CD206 + BV2 cells and CD206 + primary cultured microglia were both marked by white arrows) in BV2 cells and primary cultured microglia treated with Ctrl, LD, LT and LT + SR-717 for 24 hours. Scale bar: 100 μm. (W, X) Quantitative analysis of the percentages of CD86 + and CD206 + BV2 cells (W; n = 6 individual samples of cells per group), and primary cultured microglia (X; n = 3 individual samples of cells per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data were from at least three separate and independent studies. Arg-1: Arginase-1; CD206: cluster of differentiation 206; cGAS: cyclic GMP-AMP synthase; Ctrl: control; DMSO: dimethyl sulfoxide; IκBα: nuclear factor-κB inhibitory protein α; IL-6: interleukin-6; ns: not significant; LPS: lipopolysaccharide; p: phosphorylated; STING: stimulator of interferon genes; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

Article Snippet: rabbit anti-p-NF-κB p65 , 1: 1000 , Cell Signaling Technology , 3033 , AB_331284.

Techniques: Western Blot, Incubation, Reverse Transcription, Polymerase Chain Reaction, Expressing, Immunostaining, Cell Culture, Control

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Tryp shifts microglia from M1 to M2 phenotype after spinal cord injury by targeting the cGAS/STING/NF-κB pathway. (A) Western blot analysis of cGAS and p-STING in spinal cord treated with Tryp at 3 dpi. (B) Quantitative analysis of cGAS and p-STING protein levels shown in A (normalized to β-actin, n = 6 mice per group). (C, E) Confocal laser scanning microscopy double immunostaining images (60× magnification) of cGAS (purple) and Iba1 (green) (C), and p-STING (purple) and Iba1 (green) (E) in the spinal cord lesion area of Sham + DMSO (T8–T10), Sham + Tryp (T8–T10), SCI + DMSO and SCI + Tryp mice at 3 dpi. Higher magnification panels are single optical Z sections of the areas marked by boxes. cGAS + Iba1 + cells and p-STING + Iba1 + cells were marked by white arrowheads. Scale bars, 50 μm (left) and 20 μm (enlarge). (D, F) Quantitative analysis of the percentages of cGAS + Iba1 + cells in Iba1 + cells (D, n = 3 mice per group), and p-STING + Iba1 + cells in Iba1 + cells (F, n = 3 mice per group). (G) Western blot analysis of p-p65, p65, p-IκBα and IκBα in spinal cord treated with Tryp at 7 dpi. (H) Quantitative analysis of p-p65 (normalized to p65), p65 (normalized to β-actin), p-IκBα (normalized to IκBα) and IκBα (normalized to β-actin) protein levels shown in G ( n = 10 mice per group). (I, K) Confocal laser scanning microscopy double immunostaining images (60× magnification) of CD86 (red) and CD68 (green) (I), and CD206 (red) and CD68 (green) (K) in the spinal cord lesion area of Sham + DMSO (T8–T10), Sham + Tryp (T8–T10), SCI + DMSO and SCI + Tryp mice at 7 dpi. Higher magnification panels are single optical Z sections of the areas marked by boxes. CD86 + CD68 + cells and CD206 + CD68 + cells were marked by white arrowheads. Scale bars: 50 μm (left) and 20μm (enlarge). (J, L) Quantitative analysis of the percentages of CD68 + /DAPI (left) and CD86 + CD68 + /CD68 + (right) (J, n = 6 mice per group), and CD68 + /DAPI (left) and CD206 + CD68 + /CD68 + (right) (L, n = 6 mice per group). Data were analyzed by one-way analysis of variance with Tukey’s multiple comparisons test (left) and unpaired two-tailed Student’s t -test (right). (M) Western blot analysis of CD206 and CD86 expression in spinal cord treated with Tryp at 7 dpi. (N, O) Quantitative analysis of CD206 (N) and CD86 (O) protein levels shown in M (normalized to β-actin, n = 9 mice per group). (P–T) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (P), IL-1 β (Q), TNF-α (R), CD206 (S) and Arg-1 (T) gene expression levels in spinal cords of Sham + DMSO, Sham + Tryp, SCI + DMSO and SCI + Tryp mice at 7 dpi (normalized to β-actin, n = 6 mice per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data were from at least three separate and independent studies. Arg-1: Arginase-1; CD206: cluster of differentiation 206; cGAS: cyclic GMP-AMP synthase; DMSO: dimethyl sulfoxide; dpi: days post-injury; Iba1: ionized calcium-binding adapter molecule 1; IL-6: interleukin-6; ns: not significant; NF-κB: nuclear factor-κB; ns: not significant; p: phosphorylated; p-STING: phosphorylated stimulator of interferon genes; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

Article Snippet: rabbit anti-p-NF-κB p65 , 1: 1000 , Cell Signaling Technology , 3033 , AB_331284.

Techniques: Western Blot, Confocal Laser Scanning Microscopy, Double Immunostaining, Two Tailed Test, Expressing, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Binding Assay

Journal: Sexual Medicine

Article Title: Yimusake ameliorates corporal endothelial dysfunction by down-regulating the NLRP3 inflammasome–mediated NF-κB signaling pathway and inhibiting oxidative stress

doi: 10.1093/sexmed/qfaf079

Figure Lengend Snippet: Yimusake can inhibit the expression of the NLRP3 inflammasome-mediated NF-κB signaling pathway in vitro . (A) Western blotting was used to determine the expression of the NLRP3, p65, p-p65, IκBα, p-IκBα, and IL-18 protein expression in CCECs of each group. (B) Relative expression of NLRP3. (C) Relative expression of p-p65/p65. (D) Relative expression of p-IκBα/IκBα. (E) Relative expression of IL-18 (F) PCR detection of NLRP3 mRNA expression in CCECs. (G) RT-qPCR detection of p65 mRNA expression in CCECs, * P < .05 compared with the NC group, # P < .05 compared with the HG group, & P < .05 compared with the Sh-NLRP3 group, $ P < .05 compared with the Y group ( P < .05).

Article Snippet: Primary antibodies including rabbit anti-NF-κB p65 (1:100, BA0610, Boster, China) were added and incubated overnight at 4 °C.

Techniques: Expressing, In Vitro, Western Blot, Quantitative RT-PCR

Journal: Sexual Medicine

Article Title: Yimusake ameliorates corporal endothelial dysfunction by down-regulating the NLRP3 inflammasome–mediated NF-κB signaling pathway and inhibiting oxidative stress

doi: 10.1093/sexmed/qfaf079

Figure Lengend Snippet: Yimusake can inhibit the expression of the NLRP3 inflammasome-mediated NF-κB signaling pathway in vivo. (A) Immunohistochemical detection of NF-κB p65 protein expression in rat penile tissue (x200). (B) Relative expression of p65. (C) Western blotting was used to determine the expression of the p65, p-p65, IκBα, p-IκBα, and IL-18 protein expression in rat penile tissue of each group. (D) Relative expression of p-p65/p65. (E) Relative expression of p-IκBα/IκBα, (F) relative expression of IL-18. (G) RT-qPCR detection of p65 mRNA expression in rat penile tissue, * P < .05 compared with the NC group, # P < .05 compared with the DMED group, & P < .05 compared with the MCC950 group, $ P < .05 compared with the Y group.

Article Snippet: Primary antibodies including rabbit anti-NF-κB p65 (1:100, BA0610, Boster, China) were added and incubated overnight at 4 °C.

Techniques: Expressing, In Vivo, Immunohistochemical staining, Western Blot, Quantitative RT-PCR