9272 polyclonal rabbit anti mouse  (Cell Signaling Technology Inc)

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

Article Title: Insulin Resistance in Macrophages Alters Their Metabolism and Promotes an M2-Like Phenotype.

doi: 10.4049/jimmunol.1800065

Figure Lengend Snippet: FIGURE 1. Macrophages become insulin resistant and do not uptake glucose upon stimulation. (A) Western blot analysis of p-Akt (Ser473) in nonstimulated and after 30 min of stimulation with 100 nM insulin for control and macrophages from short-term HFD–fed mice. Summary graph of average phosphorylation of Akt in all conditions (basal and after insulin stimulation) normalized to total Akt. (B) Expression of IR in control and macrophages from short-term HFD–fed mice, measured by real-time PCR and Western blot. Akt2 macrophages are insulin resistant, as demonstrated by (C) Western blot analysis of Akt phosphorylation for WT (control) and Akt22/2 macrophages before and after insulin stimulation and (D) Akt2 phosphorylation p-Akt2 (Ser474) and (E) Akt1 phosphorylation p-Akt1 (Ser473) in macrophages from control and HFD-fed mice before and after 30 min of stimulation with 100 nM insulin. All graphs are representative of three to six independent experiments and show mean 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001, insulin stimulation versus no stimulation. #p , 0.05, ##p , 0.01, ###p , 0.001, insulin-resistant versus control macrophages.

Article Snippet: Briefly, after blocking with 5% BSA PBS (pH 7.4) for an hour at room temperature, the membranes were incubated with rabbit polyclonal anti-mouse p-Akt (Ser473) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-Akt2 (Ser474) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt2 Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-S6 (235/236) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p–4E-BP1(Thr37/46) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse inducible NO synthase (iNOS) Ab (Abcam), goat polyclonal anti-mouse arginase 1 (Abcam), goat polyclonal anti-mouse Fizz1 Ab (Abcam), rabbit polyclonal anti-mouse IGF-1Rb Ab (Cell Signaling Technology), rabbit monoclonal anti-mouse IR-b (Cell Signaling Technology), or mouse monoclonal anti-mouse b-actin (Abcam) at 4 ̊C overnight.

Techniques: Western Blot, Control, Phospho-proteomics, Expressing, Real-time Polymerase Chain Reaction

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

Article Title: Insulin Resistance in Macrophages Alters Their Metabolism and Promotes an M2-Like Phenotype.

doi: 10.4049/jimmunol.1800065

Figure Lengend Snippet: FIGURE 2. mTORC1 pathway is more active in insulin-resistant macrophages. Western blot analysis of p-S6 (Ser235/236) and p–4E-BP1 (Thr37/46) in macrophages from Akt22/2 (A) and from short-term HFD–fed mice (B) compared with control before and after 30 min of stimulation with 100 nM insulin. Evaluation of Akt2 phosphorylation (C) and mTORC1 pathway activation (D) by Western blot analysis in control and Igf1R2/2 macrophages under basal conditions and after 30 min of stimulation with 100 nM insulin. (E) Western blot analysis of Akt phosphorylation in WT (control) and Igf1R2/2 macrophages. (F) Western blot analysis of Akt2 phosphorylation in control, HFD, Igf1R2/2, and Akt22/2 macrophages before and after 30 min of stimulation with 2.6 nM IGF1. (G) Akt1 is activated in insulin-resistant macrophages. Western blot analysis of p-Akt1 (Ser473) for control, HFD, Akt22/2 Igf1R2/2, and Akt12/2 macrophages under basal conditions. All graphs are representative of three to six independent experiments and show mean 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001, insulin or IGF1 stimulation versus no stimulation. #p , 0.05, ##p , 0.01, ###p , 0.001, insulin-resistant versus control macrophages.

Article Snippet: Briefly, after blocking with 5% BSA PBS (pH 7.4) for an hour at room temperature, the membranes were incubated with rabbit polyclonal anti-mouse p-Akt (Ser473) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-Akt2 (Ser474) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse Akt2 Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p-S6 (235/236) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse p–4E-BP1(Thr37/46) Ab (Cell Signaling Technology), rabbit polyclonal anti-mouse inducible NO synthase (iNOS) Ab (Abcam), goat polyclonal anti-mouse arginase 1 (Abcam), goat polyclonal anti-mouse Fizz1 Ab (Abcam), rabbit polyclonal anti-mouse IGF-1Rb Ab (Cell Signaling Technology), rabbit monoclonal anti-mouse IR-b (Cell Signaling Technology), or mouse monoclonal anti-mouse b-actin (Abcam) at 4 ̊C overnight.

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