Journal: Immunology innovation

Article Title: Decreased signal transducers and activators of transcription (STAT) protein expression in lymphatic organs during EAE development in mice

doi: 10.7243/2053-213X-1-3

Figure Lengend Snippet: Protein was extracted from spleens isolated from EAE mice 12 days post-immunization. (A) Western blot of protein extracts demonstrates expression of STAT family members in spleens of EAE (lanes 1–4, n=4) and control mice (lanes 5–9, n=5). Expression was analyzed in both cytosolic (left column) and nuclear (right column) fractions of protein extracts. β-actin expression was also quantified in each lane. (B) Densitometry analysis of Western blots was used to quantify STAT protein expression, presented as a ratio of the density of the STAT protein band to the β-actin band. There was a significant decrease (*P<0.05; **P<0.01) of STAT1, STAT3, STAT4, STAT5 and STAT6 protein levels in both cytosolic (top row) and nuclear (bottom row) fractions of protein extracts from the spleens of EAE mice as compared with spleens from control mice. In contrast, STAT2 expression did not change in either cytosolic or nuclear extracts of spleen after EAE induction. Values are presented as mean ± SEM.

Article Snippet: All rabbit anti-mouse STAT antibodies (STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6) were purchased from Santa Cruz (Dallas, TX) and used at a dilution of 1:200 for both Western blot and immunohistochemical analysis, except for STAT1, for which a dilution of 1:10,000 was used.

Techniques: Isolation, Western Blot, Expressing, Control

Journal: Immunology innovation

Article Title: Decreased signal transducers and activators of transcription (STAT) protein expression in lymphatic organs during EAE development in mice

doi: 10.7243/2053-213X-1-3

Figure Lengend Snippet: Protein was extracted from spleens isolated from EAE mice 5 days post-immunization. (A) Western blot of protein extracts demonstrates expression of STAT family members in spleens of EAE (lanes 1–5, except cytosolic STAT 3 and 4, for which only lanes 1–4 are from EAE mice, n=4–5) and control (lanes 6–11 [lanes 5–9 for cytosolic STAT 3 and 4], n=5–6) mice. Expression was analyzed in both cytosolic (left column) and nuclear (right column) fractions of protein extracts. (B) There was a significant decrease (**P<0.01) of both cytosolic (top row) and nuclear (bottom row) STAT4 and STAT5 expression in spleens from EAE mice compared with spleens from control mice, while STAT3 and STAT6 only significantly decreased (**P<0.01) in the nuclear fraction of protein extracts from EAE mice, and remained the same in the cytosolic fractions (P>0.05). The level of STAT1 protein did not change in either cytosolic or nuclear extracts of the EAE mice compared with controls. Values are presented as mean ± SEM.

Article Snippet: All rabbit anti-mouse STAT antibodies (STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6) were purchased from Santa Cruz (Dallas, TX) and used at a dilution of 1:200 for both Western blot and immunohistochemical analysis, except for STAT1, for which a dilution of 1:10,000 was used.

Techniques: Isolation, Western Blot, Expressing, Control

Journal: Immunology innovation

Article Title: Decreased signal transducers and activators of transcription (STAT) protein expression in lymphatic organs during EAE development in mice

doi: 10.7243/2053-213X-1-3

Figure Lengend Snippet: Protein was extracted from thymuses isolated from EAE mice 5 days post-immunization. The density of bands on Western blots of thymic protein extracts are shown as a ratio of the STAT1, STAT3, STAT4, STAT5 or STAT6 band density to the β-actin band density. Thymic protein extracts from EAE (n=5) and control (n=6) mice were separated into cytosolic (left column) and nuclear (right column) fractions. Expression levels of all five STAT proteins in either cytosolic or nuclear extracts from EAE mice were equivalent to levels in control mice (P>0.05). Values are presented as mean ± SEM.

Article Snippet: All rabbit anti-mouse STAT antibodies (STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6) were purchased from Santa Cruz (Dallas, TX) and used at a dilution of 1:200 for both Western blot and immunohistochemical analysis, except for STAT1, for which a dilution of 1:10,000 was used.

Techniques: Isolation, Western Blot, Control, Expressing

Journal: Immunology innovation

Article Title: Decreased signal transducers and activators of transcription (STAT) protein expression in lymphatic organs during EAE development in mice

doi: 10.7243/2053-213X-1-3

Figure Lengend Snippet: Protein was extracted from spleens isolated from EAE mice 10 days post-immunization. (A) Western blot of protein extracts demonstrates expression of STAT family members in spleens of EAE (lanes 1–5, n=5) and control (lanes 6–11, n=6) mice. Expression was analyzed in both cytosolic (left column) and nuclear (right column) fractions of protein extracts. (B) There was a significant decrease (**P<0.01) of both cytosolic (top row) and nuclear (bottom row) expression of STAT1, STAT4, STAT5 and STAT6 proteins in spleens from EAE mice compared with spleens from control mice, while STAT3 only significantly decreased (**P<0.01) in the nuclear fraction of protein extracts from EAE mice. Values are presented as mean ± SEM.

Article Snippet: All rabbit anti-mouse STAT antibodies (STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6) were purchased from Santa Cruz (Dallas, TX) and used at a dilution of 1:200 for both Western blot and immunohistochemical analysis, except for STAT1, for which a dilution of 1:10,000 was used.

Techniques: Isolation, Western Blot, Expressing, Control

Journal: Immunology innovation

Article Title: Decreased signal transducers and activators of transcription (STAT) protein expression in lymphatic organs during EAE development in mice

doi: 10.7243/2053-213X-1-3

Figure Lengend Snippet: Representative sections cut from spleen (A–F, top row) and thymus (G–L, bottom row) of control mice were labeled by immunohistochemistry with antibodies to STAT1 (A and G), STAT2 (B and H), STAT3 (C and I), STAT4 (D and J), STAT5 (E and K) and STAT6 (F and L). The dark brown nuclear stain represents STAT antibodies binding to each specific STAT protein. In spleen (A–F), all six STAT proteins were mainly localized in the T cell area of the white pulp. Immunostaining of all STAT proteins localized within cell nuclei in the spleen (A–F). In the thymus (G–L), immunostaining for all STAT proteins was also confined to the nuclei, except STAT2 (H), which was localized in both cytoplasm and nuclei. Magnification=400×.

Article Snippet: All rabbit anti-mouse STAT antibodies (STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6) were purchased from Santa Cruz (Dallas, TX) and used at a dilution of 1:200 for both Western blot and immunohistochemical analysis, except for STAT1, for which a dilution of 1:10,000 was used.

Techniques: Control, Labeling, Immunohistochemistry, Staining, Binding Assay, Immunostaining

Journal: Immunology innovation

Article Title: Decreased signal transducers and activators of transcription (STAT) protein expression in lymphatic organs during EAE development in mice

doi: 10.7243/2053-213X-1-3

Figure Lengend Snippet: Representative sections cut from spleens isolated on day 12 post-immunization from control (A–G, top row) and EAE (H–N, bottom row) mice were labeled by immunohistochemistry with antibodies to STAT1 (A and H), STAT2 (B and I), STAT3 (C and J), STAT4 (D and K), STAT5 (E and L) and STAT6 (F and M). In EAE mice, numerous secondary lymphatic follicles formed and were scattered across the spleen. The dark brown immunostaining formed by STAT antibodies binding to each specific STAT protein was confined within the follicles, except STAT2 staining, which was localized in other areas of the spleen, but not in the follicles. The numbers of positively stained cells for all splenic STAT proteins were higher in control mice than in EAE mice. The intensity of the immunostaining for all splenic STAT proteins was also stronger in control mice than in EAE mice. G and N are negative controls in which the primary antibody was replaced by normal serum. Magnification=40×.

Article Snippet: All rabbit anti-mouse STAT antibodies (STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6) were purchased from Santa Cruz (Dallas, TX) and used at a dilution of 1:200 for both Western blot and immunohistochemical analysis, except for STAT1, for which a dilution of 1:10,000 was used.

Techniques: Isolation, Control, Labeling, Immunohistochemistry, Immunostaining, Binding Assay, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Myeloid Deletion of Cdc42 Protects Liver From Hepatic Ischemia-Reperfusion Injury via Inhibiting Macrophage-Mediated Inflammation in Mice

doi: 10.1016/j.jcmgh.2024.01.023

Figure Lengend Snippet: Myeloid deletion of Cdc42 enhances the M2-type differentiation of macrophages via regulating STATs signaling. THP1 cells were pretreated with PMA (100 ng/mL) and ML141 (10 μM) for differentiation and then stimulated with LPS (100 ng/mL) and IFN-γ (20 ng/mL) for 1 hour for M1 induction, or IL4 and IL13 (both at 40 ng/mL) for 48 hours for M2 induction. The images ( A ) and quantitative results ( C–F ) of the phosphorylation of STAT1, STAT3, and STAT6 and expressions of SOCS3 were detected by Western blot analysis in THP1 cells, n = 3. The images ( B ) and quantitative results ( G–J ) of the phosphorylation of STAT1, STAT3, and STAT6 and expressions of SOCS3 were measured by Western blot analysis in BMDMs stimulated with LPS (100 ng/mL) for 3 hours or IL4 (40 ng/mL) for 48 hours, respectively, n = 3. n. s., no significance; ∗ P < .05; ∗∗∗ P < .001.

Article Snippet: The proteins were extracted and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare), which will be blocked with 5% BSA or skimmed milk in Tris-buffered saline containing 0.1% Tween 20 and probed with antibodies against iNOS (Proteintech, 22226-1-AP), ARG1 (Proteintech, 66129-1-AP), IL10 (proteintech,60269-1-Ig), IL6 (CST, 12912S), SOCS3 (Proteintech, 14025-1-AP), STAT Antibody Sampler Kit (CST, 9939T), Phospho-STAT Antibody Sampler (CST, 9914T: Phospho-STAT1 Tyr701 and Phospho-STAT3 Tyr705, Phospho-STAT6 Tyr641), respectively.

Techniques: Western Blot