Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Glycogen synthase kinase-3beta facilitates IFN-gamma-induced STAT1 activation by regulating Src homology-2 domain-containing phosphatase 2.

doi: 10.4049/jimmunol.0804033

Figure Lengend Snippet: FIGURE 2. IFN- regulates Akt and Pyk2, which then activates GSK-3 and inflammation. A and C, RAW264.7 cells were treated with IFN- for the indicated time periods. We used Western blotting to determine phosphoryla- tion of GSK-3 (Ser9 and Tyr216) and Akt (Ser473). B, D, and F, With or without BIO, OA, or Sph-24 pretreatment for 0.5 h, RAW264.7 cells were then treated with IFN-. Western blotting was used to determine phosphory- lation of GS (Ser641) in cells 2 h posttreatment, and Akt (Ser473) and GSK-3 (Ser9) in cells 0.5 h posttreatment. E, RAW264.7 cells were treated with IFN- for the indicated time periods. We used a kit to determine SMase activity. Percentages relative to those of the control are shown. G, Western blotting was used to determine the time kinetics of phosphorylation of Pyk2 (Tyr402) in IFN--stimulated RAW264.7 cells. H–J, With or without A9 pretreatment for 0.5 h, RAW264.7 cells were then treated with IFN-. Western blotting was used to determine phosphorylation of GSK-3 (Ser9) 0.25 h posttreatment and GSK-3 (Tyr216) 2 h posttreatment. Griess reagent was used to determine NO production 24 h posttreatment. TNF- production was determined using ELISA 6 h posttreatment. For Western blot analysis, -actin was the internal control. Data shown are representative of three individual experiments. For the activity assay, nitrite detection, and ELISA, the data are means SD obtained from three individual cultures. , p 0.05 compared with the untreated or the IFN- group.

Article Snippet: Abs against phospho-GSK-3 (Ser9), phospho-GSK-3 (Tyr216), GSK-3 / , phospho-GS (Ser641), GS, phospho-Akt (Ser473), Akt, phospho-Pyk2 (Tyr402), Pyk2, phospho-STAT1 (Tyr701), phospho-STAT1 (Ser727), STAT1, phospho-Jak2 (Tyr1007/1008), Jak2, phospho-Src (Tyr416), Src, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-SHP2 (Tyr542), SHP2, SOCS1, and SOCS3 were purchased from Cell Signaling Technology.

Techniques: Western Blot, Activity Assay, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Glycogen synthase kinase-3beta facilitates IFN-gamma-induced STAT1 activation by regulating Src homology-2 domain-containing phosphatase 2.

doi: 10.4049/jimmunol.0804033

Figure Lengend Snippet: FIGURE 3. IFN- induces PC-PLC-, PKC-, and Src-regulated GSK-3 activation and inflammation. RAW264.7 cells were pretreated with D609, Go¨6976, or PP1 for 0.5 h and then treated with IFN-. We used Western blotting to determine the phosphorylation of Pyk2 (Tyr402) and GSK-3 (Tyr216) 2 h posttreatment and GSK-3 (Ser9) 0.5 h posttreatment and iNOS expression 24 h posttreatment (A–C). Griess reagent and ELISA were used to determine NO (D) 24 h posttreatment and TNF- (E) pro- duction 6 h posttreatment. For Western blot analysis, -actin was the in- ternal control. Data shown are representative of three individual experi- ments. The OD of Pyk2 (Tyr402)/total Pyk2 has been clarified using ImageJ software. For nitrite detection and ELISA, the data are means SD ob- tained from three individual cultures. , p 0.05 compared with the IFN- group.

Article Snippet: Abs against phospho-GSK-3 (Ser9), phospho-GSK-3 (Tyr216), GSK-3 / , phospho-GS (Ser641), GS, phospho-Akt (Ser473), Akt, phospho-Pyk2 (Tyr402), Pyk2, phospho-STAT1 (Tyr701), phospho-STAT1 (Ser727), STAT1, phospho-Jak2 (Tyr1007/1008), Jak2, phospho-Src (Tyr416), Src, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-SHP2 (Tyr542), SHP2, SOCS1, and SOCS3 were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Control, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Glycogen synthase kinase-3beta facilitates IFN-gamma-induced STAT1 activation by regulating Src homology-2 domain-containing phosphatase 2.

doi: 10.4049/jimmunol.0804033

Figure Lengend Snippet: FIGURE 4. IFN- induces Jak2-regulated activation of PC-PLC, Pyk2, and GSK-3 independently of IFNGR1. A, Splenocytes harvested from IFNGR1 wild-type (IFNGR1 WT) and IFNGR1-deficient (IFNGR1/) C57BL/6 mice were treated with IFN- for 0.5 h. We used a kit to deter- mine the activity of PC-PLC. Percentages relative to those of the control are shown. B, Western blotting was used to determine phosphorylation of STAT1 (Tyr701) 1 h posttreatment and Pyk2 (Tyr402) and GSK-3 (Tyr216) 3 h posttreatment. C, RAW264.7 cells were pretreated with AG490 for 0.5 h and then treated with IFN- for 0.5 h. The activity of PC-PLC was determined using a kit. Percentages relative to those of the control are shown. D, Western blotting was used to determine phosphorylation of STAT1 (Tyr701) and Jak2 (Tyr1007/1008) 1 h posttreatment and Pyk2 (Tyr402) and GSK-3 (Tyr216) 3 h posttreatment. For the activity assay, the data are means SD obtained from three individual cultures. , p 0.05 compared with the untreated or the IFN- group. For Western blot analysis, -actin was the internal control. Data shown are representative of three individual experiments.

Article Snippet: Abs against phospho-GSK-3 (Ser9), phospho-GSK-3 (Tyr216), GSK-3 / , phospho-GS (Ser641), GS, phospho-Akt (Ser473), Akt, phospho-Pyk2 (Tyr402), Pyk2, phospho-STAT1 (Tyr701), phospho-STAT1 (Ser727), STAT1, phospho-Jak2 (Tyr1007/1008), Jak2, phospho-Src (Tyr416), Src, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-SHP2 (Tyr542), SHP2, SOCS1, and SOCS3 were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Activity Assay, Control, Western Blot, Phospho-proteomics

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Reversal of maladaptive fibrosis and compromised ventricular function in the pressure overloaded heart by a caveolin-1 surrogate peptide

doi: 10.1038/labinvest.2016.153

Figure Lengend Snippet: Antibodies used in experiments

Article Snippet: Membranes were blocked, incubated with primary antibodies , washed, incubated with HRP-labeled secondary antibodies, and detected by enhanced chemiluminescence. table ft1 table-wrap mode="anchored" t5 caption a7 Flow cytometry Specificity (description) Label Product # Concentration CD45 (Rat anti-mouse, clone 30-F11) PE-Cy7 BD 552848 0.50 μ g/ml Collagen Iα1 C-terminal propeptide (Rabbit anti-human, polyclonal) None Ref. 75 1.67 μ g/ml CD11b (Rat anti-mouse, clone M1/70) AF647 BD 557686 1 μ g/ml CD16 (Rat anti-mouse, clone 2.4G2) APC BD 558636 1 μ g/ml CD68 (Rat anti-mouse, clone FA-11) APC BioLegend 137008 1 μ g/ml HSP47 (Mouse anti-rat, clone M16.10A1) AF647 a Enzo ADI-SPA-70 1 μ g/ml Western blotting Specificity (description) Product # Collagen Iα1 C-terminal telopeptide (Rabbit anti-human, polyclonal) Ref. 75 HSP47 (Mouse anti-rat, clone M16.10A1) Enzo ADI-SPA-70 β 1-integrin (Rabbit anti-human, polyclonal) Cell signaling 4706 β 3-integrin (Rabbit anti-human/mouse, polyclonal) Cell signaling 4702 Pky2 pTyr402 (Rabbit anti-human, polyclonal) Cell signaling 3291 Src pTyr416 (Rabbit anti-human, polyclonal) Cell signaling 2101 GAPDH (Mouse anti-human, clone 6C5) Fitzgerald 10R-G109A Actin (Rabbit polyclonal) Sigma-Aldrich A2066 eNOS (Rabbit anti-human monoclonal) Cell signaling 32027 eNOS pSer1177 (Rabbit anti-human polyclonal) Cell signaling 9571 Open in a separate window Abbreviation: AF, Alexa Fluor. a Labeled using an Alexa Fluor 647 Protein Labeling Kit (Invitrogen, Carlsbad, CA, A-20173) as described by the manufacturer.

Techniques: Cytometry, Concentration Assay, Western Blot