Journal: Regenerative biomaterials

Article Title: The polycaprolactone/silk fibroin/carbonate hydroxyapatite electrospun scaffold promotes bone reconstruction by regulating the polarization of macrophages.

doi: 10.1093/rb/rbac035

Figure Lengend Snippet: Figure 8. The PCL/SF/CHA scaffold activated the JAK/STAT5 pathway and inhibited the AKT and NF-jB pathways in macrophages. (A and B) Western blot of JAK/STAT5 pathway-associated proteins (A) and AKT and p65 pathway-associated proteins (B) in RAW 264.7 cells cultured on scaffolds. (C) The expression of Mrc1 and IL-10 in RAW 264.7 cells. RAW 264.7 cells were pretreated by JAK/STAT5 inhibitor SH-4-54 (10 lM) and then cultured on the scaffolds. (D and E) The expression of iNOS and IL-1b in RAW 264.7 cells. RAW 264.7 cells were pretreated by 10 lM AKT inhibitor MK2206 (D) or 10 lM NF-jB inhibitor Bay-11-7028 (E) and then cultured on the scaffolds. The P values represented by *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The membranes were blocked by 5% skim milk (g/mL) or 5% BSA (g/mL) for 60 min at room temperature and then incubated with primary antibodies against p-AKT (1:2000, CST, 9271S), AKT (1:1000, CST, 4691 T), p-p65 (1:1000, CST, 3033 T), p65 (1:1000, CST, 8242 T), b-actin (1:10000, CST, 58169S), STAT5 (1:1000, CST, 25656S), p-STAT5 (1:1000, CST, 9314S), p-JAK1 (1:1000, CST, 74129 T), p-JAK2 (1:1000, CST, 4406 T) and p-JAK3 (1:1000, CST, 5031 T) at 4 C overnight, followed by secondary antibodies for 1 h at room temperature.

Techniques: Western Blot, Cell Culture, Expressing

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling.

doi: 10.1210/me.2003-0256

Figure Lengend Snippet: Fig. 3. JAK2 Mutants Studied and Summary of Results Diagram of JAK2 mutants previously studied (20) (JAK2-1–999 and JAK2-1–239) and those newly prepared for the current study. Each mutant is named so as to indicate the remaining JAK2 residues, the residues deleted (), or the residues substituted (as for JAK2FAAAA), as detailed in Materials and Methods. The FERM, kinase-like, and kinase domains are indicated. The ability of the indicated mutants to allow GH-induced JAK2 tyrosine phosphorylation, STAT5 tyrosine phosphorylation, and/or Spi2.1 trans-activation in Figs. 4–8 is summarized ( or ).

Article Snippet: Mouse anti-STAT5 monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Mutagenesis, Phospho-proteomics, Activation Assay

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling.

doi: 10.1210/me.2003-0256

Figure Lengend Snippet: Fig. 7. Comparison of JAK2-FAAAA with WT JAK2 A and B, GH-induced tyrosine phosphorylation of JAK2 and STAT5. A, 2A-rbGHR cells transiently transfected with either WT JAK2 (lanes 1 and 2) or JAK2-FAAAA (lanes 3 and 4) were serum-starved and treated with () or without () GH for 15 min before detergent lysis and anti-JAK2AL33 immunoprecipitation. Precipitated proteins were resolved by SDS-PAGE and immunoblotted sequentially with anti-pY (upper panel) and anti-JAK2AL33 (lower panel). The data shown are representative of two such experiments. B, 2A-rbGHR cells transiently transfected a STAT5b expression vector (lanes 1–6) along with WT JAK2 (lanes 3 and 4), JAK2-FAAAA (lanes 5 and 6), or empty vector (lanes 1 and 2) were serum-starved and treated with () or without () GH for 10 min before detergent lysis. Proteins were resolved by SDS-PAGE and immunoblotted with anti-pSTAT5 antibody. The data shown are representative of two such experiments. C, GH-induced Spi2.1 trans-activation. JAK2-FAAAA was analyzed for reporter gene trans-activation as described in Fig. 4. Samples were exposed to varying concentrations of GH for 18 h before measurement of luciferase activity as described in Materials and Methods. Data are plotted as the fold increase in activity at each concentration relative to untreated samples (mean SE of triplicate determinations).

Article Snippet: Mouse anti-STAT5 monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Comparison, Phospho-proteomics, Transfection, Lysis, Immunoprecipitation, SDS Page, Expressing, Plasmid Preparation, Activation Assay, Luciferase, Activity Assay, Concentration Assay

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling.

doi: 10.1210/me.2003-0256

Figure Lengend Snippet: Fig. 8. Functional Complementation between JAK2 Mutants A–C, GH-induced STAT5 tyrosine phosphorylation and Spi2.1 trans-activation. A, 2A-rbGHR cells transiently transfected with STAT5b (lanes 1–8) along with WT JAK2 (lanes 1 and 2), JAK2-1–47 (lanes 3 and 4), JAK2-1–999 (lanes 5 and 6), or JAK2-1–47 plus JAK2-1–999 (lanes 7 and 8) were serum-starved and treated with () or without () GH for 10 min before detergent lysis. Proteins were resolved by SDS-PAGE and immunoblotted sequentially with anti-pSTAT5 antibody (upper panel) and anti-STAT5 (lower panel). The data shown are representative of three such experiments. B and C, JAK2-1–47 plus JAK2-1–999 (B) and JAK2-1–239 plus JAK2-1–511 (C) were analyzed for reporter gene trans-activation as described in Figs. 5 and 6. Data are plotted as the GH-induced fold increase in activity (mean SE; n 3 independent experiments in B and C), using that mediated by WT JAK2 within each experiment as 100%.

Article Snippet: Mouse anti-STAT5 monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Functional Assay, Phospho-proteomics, Activation Assay, Transfection, Lysis, SDS Page, Activity Assay