p-rip1(ser166)  (Affinity Biosciences)

Journal: Clinical and experimental hypertension (New York, N.Y. : 1993)

Article Title: Disruption of the caspase-1/IL-1β axis alleviates myocardial Ischemia/Reperfusion injury via improvement of mitochondrial homeostasis and reduction of Pyroptosis.

doi: 10.1080/10641963.2025.2506619

Figure Lengend Snippet: Figure 4. Caspase-1 silencing reduces the secretion of IL-1β and inhibits myocardial injury due to cardiomyocyte pyroptosis. A, Western blot of caspase-1 and IL-1β proteins in myocardial tissues of I/R mice treated with sh-caspase-1#1 or sh-caspase-1#2. B, IL-1β serum levels in I/R mice treated with sh-caspase-1 measured by ELISA. C, LDH level measurement in the serum of I/R mice treated with sh-caspase-1. D, evaluation of cardiac physical function in I/R mice treated with sh-caspase-1 by echocardiography. E, myocardial infarct size in I/R mice treated with sh-caspase-1 detected by TTC staining. F, HE staining of cardiomyocyte injury in I/R mice treated with sh-caspase-1. G, cardiomyocyte pyroptosis in I/R mice treated with sh-caspase-1 evaluated by CaV3 (live cardiomyocytes, green) and EBD (necrotic cells, red) double staining. H, Western blot of RIP1, p-RIP1, RIPK3, and p-RIPK3, MLKL, and p-MLKL proteins in myocardial tissues of I/R mice treated with sh-caspase-1. I, morphological changes of myocardial cells in ultrathin sections of myocardial tissue were observed by transmission electron microscopy. n = 10 mice for each treatment. * p < .05.

Article Snippet: After 5% skimmed milk powder blocking, the membrane was probed with primary rabbit antibodies to GAPDH (ab9485, Abcam), Caspase-1 (sc -392 736, Santa Cruz Biotechnology), IL-1β (ab234437, Abcam), RIP1 (ab300617, Abcam), p-RIP1 (53286, Cell Signaling Technology, Shanghai, China), RIPK3 (ab62344, Abcam), p-RIPK3 (ab195117, Abcam), MLKL (ab255747, Abcam), p-MLKL (ab196436, Abcam) and then with secondary antibody peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (#111035003, Jackson ImmunoResearch).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Double Staining, Transmission Assay, Electron Microscopy

Journal: Clinical and experimental hypertension (New York, N.Y. : 1993)

Article Title: Disruption of the caspase-1/IL-1β axis alleviates myocardial Ischemia/Reperfusion injury via improvement of mitochondrial homeostasis and reduction of Pyroptosis.

doi: 10.1080/10641963.2025.2506619

Figure Lengend Snippet: Figure 5. Caspase-1 silencing suppresses H/R-induced cardiomyocyte pyroptosis in vitro. A-B, Western blot of caspase-1 protein in primary cardiomyocytes after 30 min of hypoxia and reoxygenation at 4, 6 and 8 h. C, viability of H/R-induced cardiomyocytes treated with sh-caspase-1 measured by CCK-8 assay. D, LDH release in H/ R-induced cardiomyocytes treated with sh-caspase-1. E, Representative images of pyroptosis of H/R-induced cardiomyocytes treated with sh-caspase-1 measured by PI (red)/DAPI (blue) double staining. F, PI+/DAPI+ H/R-induced cardiomyocytes treated with sh-caspase-1. G, Western blot of RIP1, p-RIP1, RIPK3, and p-RIPK3, MLKL, and p-MLKL proteins in H/R-induced cardiomyocytes treated with sh-caspase-1. Cell experiments were repeated at least three times independently. * p < .05.

Article Snippet: After 5% skimmed milk powder blocking, the membrane was probed with primary rabbit antibodies to GAPDH (ab9485, Abcam), Caspase-1 (sc -392 736, Santa Cruz Biotechnology), IL-1β (ab234437, Abcam), RIP1 (ab300617, Abcam), p-RIP1 (53286, Cell Signaling Technology, Shanghai, China), RIPK3 (ab62344, Abcam), p-RIPK3 (ab195117, Abcam), MLKL (ab255747, Abcam), p-MLKL (ab196436, Abcam) and then with secondary antibody peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (#111035003, Jackson ImmunoResearch).

Techniques: In Vitro, Western Blot, CCK-8 Assay, Double Staining

Journal: Clinical and experimental hypertension (New York, N.Y. : 1993)

Article Title: Disruption of the caspase-1/IL-1β axis alleviates myocardial Ischemia/Reperfusion injury via improvement of mitochondrial homeostasis and reduction of Pyroptosis.

doi: 10.1080/10641963.2025.2506619

Figure Lengend Snippet: Figure 7. IL-1β participates in the cardiomyocyte pyroptosis due to mitochondrial dysfunction by caspase-1. A, immunofluorescence staining of the co-localization of caspase-1 and IL-1β in the H/R-induced cardiomyocytes. B, Western blot of IL-1β protein in H/R-induced cardiomyocytes treated with sh-caspase-1. C, IL-1β levels in the supernatant of H/R-induced cardiomyocytes treated with sh-caspase-1 measured by ELISA. D, viability of H/R-induced cardiomyocytes treated with sh-caspase-1 measured by CCK-8, E, LDH release in H/R-induced cardiomyocytes treated with sh-caspase-1. F, Representative images of pyroptosis of H/R-induced cardiomyocytes treated with sh-caspase-1 measured by PI (red)/DAPI (blue) double staining. G, PI+/DAPI+ H/R-induced cardiomyocytes treated with sh-caspase-1. H, Western blot of RIP1, p-RIP1, RIPK3, and p-RIPK3, MLKL, and p-MLKL proteins in H/R-induced cardiomyocytes treated with sh-caspase-1. Cell experiments were repeated at least three times independently. * p < .05.

Article Snippet: After 5% skimmed milk powder blocking, the membrane was probed with primary rabbit antibodies to GAPDH (ab9485, Abcam), Caspase-1 (sc -392 736, Santa Cruz Biotechnology), IL-1β (ab234437, Abcam), RIP1 (ab300617, Abcam), p-RIP1 (53286, Cell Signaling Technology, Shanghai, China), RIPK3 (ab62344, Abcam), p-RIPK3 (ab195117, Abcam), MLKL (ab255747, Abcam), p-MLKL (ab196436, Abcam) and then with secondary antibody peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (#111035003, Jackson ImmunoResearch).

Techniques: Immunofluorescence, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Double Staining

Journal: Redox biology

Article Title: Deficiency of muscle-generated brain-derived neurotrophic factor causes inflammatory myopathy through reactive oxygen species-mediated necroptosis and pyroptosis.

doi: 10.1016/j.redox.2024.103418

Figure Lengend Snippet: Fig. 4. Enhanced necroptosis in the muscle of MBKO mice. A. Necroptosis signaling in the gastrocnemius of female Fl/Fl and MBKO mice (6 months old). Quantification of the immunoblot is shown in the right panel (*: P < 0.05, **: P < 0.01, ***: P < 0.001, Student’s t-test, n = 3). B. The presence of necrosome in the gastrocnemius of female Fl/Fl and MBKO mice (6 months old) was determined by co-immunoprecipitation (top panel). The amount of RIP1 (middle panel) and RIP3 (bottom panel) input was also examined. C. Intramyocellular content of phosphorylated RIP3 in the gastrocnemius of female Fl/Fl and MBKO mice (6 months old) was examined by immunofluorescence staining. The yellow and green arrows indicate representative myofibers with high and low cellular content of phosphorylated RIP3, respectively. The scale bar represents 50 μm. D. IgG uptake in the gastrocnemius of female Fl/Fl and MBKO mice (6 months old) was determined by immunohistochemical staining. The scale bar represents 50 μm. E. Evans blue uptake in the gastrocnemius of female Fl/Fl and MBKO mice (6 months old) was determined by fluorescence microscopy. The yellow and green arrows indicate representative myofibers with ruptured and intact sarcolemma, respectively. The scale bar represents 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Antibodies against ASC (67824), cleaved-caspase 1 D296 (89332), cleaved-caspase 3 D175 (9661), caspase 3 (9662), cleaved-caspase 8 D387 (8592), p-ERK1/2 T202/Y204 (9106), ERK1/2 (9102), p-IRF3 S396 (29047), IRF3 (4302), p-JNK T183/Y185 (4668), JNK (9252), p–NF–κB p65 S536 (3033), NFκB p65 (8242), NLRP3 (15101), p-RIP1 S616 (53286), RIP1 (3493), pRIP3 T231/S232 (91702), RIP3 (15828), p-MLKL S345 (37333), MLKL (37705), and PARP (9542) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Microscopy