anti phosphor jak2 tyr1007 1008  (Cell Signaling Technology Inc)

Journal: Cells

Article Title: Necrostatin-1 Alleviates Lung Ischemia-Reperfusion Injury via Inhibiting Necroptosis and Apoptosis of Lung Epithelial Cells

doi: 10.3390/cells11193139

Figure Lengend Snippet: Necrostatin-1 inhibited IR-induced necroptosis of lung epithelial cells in mice. The mouse LIRI was induced by clamping the left pulmonary hilum of the mouse for 1 h using a microvascular clip and then removing the clip for reperfusion for 2 h. Nec-1 (1 mg/kg) was administered intraperitoneal injection 1 h before ischemia. ( A ) Western blotting of necroptosis-related proteins (RIPK1, phosphor-RIPK1, RIPK3, phosphor-RIPK3, MLKL, and phosphor-MLKL) in lung tissue homogenates. ( B – G ) Quantification of proteins from Western blots in panel A. ( H ) Immunofluorescence staining of phosphor-MLKL and SPC (a marker of lung epithelial cell) in peripheral bronchial epithelium (upper panel) and alveolar epithelium (lower panel). White arrows indicated that pMLKL was localized at airway or alveolar epithelial cells. ( I ) Percentage of SPC and pMLKL double-positive cells in SPC positive cells. ( J ) The concentration of HMGB1 in BALF detected using ELISA. ( K ) Immunohistochemical staining of phosphor-MLKL in different groups. Black arrows indicated the pMLKL-positive areas in the bronchial epithelium. ( L ) The ratio of the pMLKL-positive area to the whole lung tissue area. n = 6 animals per group. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with Sham; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with LIRI; one-way ANOVA.

Article Snippet: The following antibodies were used: mouse monoclonal antibody against RIPK1 (1:1000, ab56164, Abcam, Cambridge, UK), rabbit polyclonal antibody against RIPK3 (1:1000, ab56164, Abcam, Cambridge, UK), rabbit polyclonal antibody against phosphor-RIPK3 (S227, 1:1000, ab209384, Abcam, Cambridge, UK), rabbit polyclonal antibody against MLKL (1:1000, ab79823, Abcam, Cambridge, UK), rabbit polyclonal antibody against phosphor-MLKL (S358, 1:1000, ab187091, Abcam, Cambridge, UK), rabbit polyclonal phosphor-RIPK1 antibody (S166, 1:1000, #44590, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal antibody against Phospho-IκB (1:1000, #9246, Cell Signaling Technology, Danvers, MA, USA), rabbit Polyclonal Antibody against cleaved caspase 3 antibody (1:1000, #9661, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal antibody against IκBα (1:1000, AF5204, Beyotime Biotechnology, Shanghai, China), rabbit monoclonal antibody against Bax (#CPA1092, Cohesion Bio, London, UK), rabbit monoclonal antibody against cleaved caspase 8 (#WL00659, Wanleibio, Shenyang, China), and mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, #60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Injection, Western Blot, Immunofluorescence, Staining, Marker, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining

Journal: Cells

Article Title: Necrostatin-1 Alleviates Lung Ischemia-Reperfusion Injury via Inhibiting Necroptosis and Apoptosis of Lung Epithelial Cells

doi: 10.3390/cells11193139

Figure Lengend Snippet: Necrostatin-1 reduced CI/R-induced cell death through the inhibition of RIPK1-dependent apoptosis and necroptosis in BEAS-2B cells. BEAS-2B cells were incubated in a 4 °C chamber filled with 50% oxygen for 18 h with ice-cold lung preservation Perfadex ® solution to simulate the procedure of clinical donor lung preservation and then subjected to a warm culture medium to mimic reperfusion. Necrostatin-1 (Nec-1, 30 μM) was added to the medium at the beginning of cold-ischemia and sustained during reperfusion. ( A ) Propidium iodide (PI) and Hoechst staining in BEAS-2B cells after CI/R. ( B , C ) Quantification of necrotic and apoptotic cells in panel A. ( D ) Western blotting of necroptosis- and apoptosis-related proteins (RIPK1, phosphor-RIPK1, RIPK3, phosphor-RIPK3, MLKL, phosphor-MLKL, cleaved RIPK-1, and Cleaved caspase 3) in BEAS-2B cells. ( E – L ) Quantification of proteins from Western blots in panel D. ( M ) The colocalization of RIPK1 and RIPK3 determined by immunofluorescence staining. ( N ) Cell counting of the RIPK1 and RIPK3 double-positive cells. Three independent experiments were performed. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with Control; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with CI/R. One-way ANOVA.

Article Snippet: The following antibodies were used: mouse monoclonal antibody against RIPK1 (1:1000, ab56164, Abcam, Cambridge, UK), rabbit polyclonal antibody against RIPK3 (1:1000, ab56164, Abcam, Cambridge, UK), rabbit polyclonal antibody against phosphor-RIPK3 (S227, 1:1000, ab209384, Abcam, Cambridge, UK), rabbit polyclonal antibody against MLKL (1:1000, ab79823, Abcam, Cambridge, UK), rabbit polyclonal antibody against phosphor-MLKL (S358, 1:1000, ab187091, Abcam, Cambridge, UK), rabbit polyclonal phosphor-RIPK1 antibody (S166, 1:1000, #44590, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal antibody against Phospho-IκB (1:1000, #9246, Cell Signaling Technology, Danvers, MA, USA), rabbit Polyclonal Antibody against cleaved caspase 3 antibody (1:1000, #9661, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal antibody against IκBα (1:1000, AF5204, Beyotime Biotechnology, Shanghai, China), rabbit monoclonal antibody against Bax (#CPA1092, Cohesion Bio, London, UK), rabbit monoclonal antibody against cleaved caspase 8 (#WL00659, Wanleibio, Shenyang, China), and mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, #60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Inhibition, Incubation, Preserving, Staining, Western Blot, Immunofluorescence, Cell Counting

Journal: Cells

Article Title: Necrostatin-1 Alleviates Lung Ischemia-Reperfusion Injury via Inhibiting Necroptosis and Apoptosis of Lung Epithelial Cells

doi: 10.3390/cells11193139

Figure Lengend Snippet: Necrostatin-1 inhibited the formation of necrosome but promoted apoptosis in mouse LIRI. The mouse LIRI was induced by clamping the left pulmonary hilum of the mouse for 1 h using a microvascular clip and then removing the clip for reperfusion for 2 h. Nec-1 (1 mg/kg) was administered intraperitoneal injection 1 h before ischemia. ( A ) Western blotting of RIPK1, phosphor-RIPK1, RIPK3, and phosphor-RIPK3 in soluble parts (upper panel) and insoluble parts (lower panel) of lung tissue homogenates. n = 3 animals per group. ( B ) Western blotting of apoptosis-related proteins (cleaved RIPK1, caspase 3, cleaved caspase 3, caspase 8, cleaved caspase 8, and Bax) in lung tissue homogenates. ( C – H ) Quantification of proteins from Western blots in panel B. n = 6 animals per group. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with Sham; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with LIRI; One-way ANOVA.

Article Snippet: The following antibodies were used: mouse monoclonal antibody against RIPK1 (1:1000, ab56164, Abcam, Cambridge, UK), rabbit polyclonal antibody against RIPK3 (1:1000, ab56164, Abcam, Cambridge, UK), rabbit polyclonal antibody against phosphor-RIPK3 (S227, 1:1000, ab209384, Abcam, Cambridge, UK), rabbit polyclonal antibody against MLKL (1:1000, ab79823, Abcam, Cambridge, UK), rabbit polyclonal antibody against phosphor-MLKL (S358, 1:1000, ab187091, Abcam, Cambridge, UK), rabbit polyclonal phosphor-RIPK1 antibody (S166, 1:1000, #44590, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal antibody against Phospho-IκB (1:1000, #9246, Cell Signaling Technology, Danvers, MA, USA), rabbit Polyclonal Antibody against cleaved caspase 3 antibody (1:1000, #9661, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal antibody against IκBα (1:1000, AF5204, Beyotime Biotechnology, Shanghai, China), rabbit monoclonal antibody against Bax (#CPA1092, Cohesion Bio, London, UK), rabbit monoclonal antibody against cleaved caspase 8 (#WL00659, Wanleibio, Shenyang, China), and mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, #60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Injection, Western Blot