rabbit polyclonal anti src 2 antibody  (Abcam)

Journal: Cell reports

Article Title: Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition

doi: 10.1016/j.celrep.2021.110075

Figure Lengend Snippet: (A) Dual immunofluorescence for GFP (green) and SRC-2 (red) in the ARH of POMC-EGFP mice (controls, upper panels) and pomcSRC-2-KO/POMC-EGFP mice (lower panels). Arrowheads point to double-labeled neurons. Scale bar represents 100 μm. 3V, 3 rd ventricle; ARH, arcuate nucleus of the hypothalamus; ME, median eminence; VMH, ventromedial hypothalamic nucleus. (B and C) Changes in body weight (B), fat mass, or lean mass (C) of chow-fed male control and pomcSRC-2-KO littermates (3 to 4 months old) after a 24-h fasting. Data are presented as mean ± SEM (% baseline before fasting). n = 6 or 10 mice per group. **p < 0.01 in t tests. (D) Daily energy expenditure measured by CLAMS in male control and pomcSRC-2-KO littermates (3 months old) during ad libitum fed, fasted, and refed periods. Data are presented as mean ± SEM. n = 8 or 9 mice per group. *p < 0.05 and ***p < 0.001 in two-way ANOVA analyses repeated measurement followed by Sidak tests. (E) Representative microscopic images showing a recorded POMC neuron (GFP-labeled in POMC-EGFP mice). Scale bar represents 10 μm. (F) Representative action potential traces in POMC neurons from fasted control versus pomcSRC-2-KO mice. (G and H) Quantifications of firing rate (G) and resting membrane potential (H) in POMC neurons from fasted control versus pomcSRC-2-KO mice. Data are presented as mean ± SEM. n = 17 or 21 neurons from 3 mice per group. *p < 0.05 in t tests. (I) Representative traces for mIPSC recorded in POMC neurons from fasted control versus pomcSRC-2-KO mice. (J and K) Quantifications of frequency (J) and amplitude (K) of mIPSC. Data are presented as mean ± SEM. n = 15 or 17 neurons from 3 mice per group. *p < 0.05 in t tests. (L) Relative mRNA levels of indicated genes measured in POMC neurons isolated from control versus pomcSRC-2-KO mice. Data are presented as mean ± SEM. n = 8 samples per group. *p < 0.05 in t tests.

Article Snippet: The proteins were analyzed by immunoblotting with rabbit monoclonal anti-FoxO1 antibody (1:3,000, #2880, Cell Signaling) or mouse monoclonal anti-FoxO1 antibody (1:3,000, #97635, Cell Signaling), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was rabbit anti-mouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Techniques: Immunofluorescence, Labeling, Isolation

Journal: Cell reports

Article Title: Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition

doi: 10.1016/j.celrep.2021.110075

Figure Lengend Snippet: (A) Effects of SRC-2 on POMC-luciferase activity in “fed” and fasted cells. Data are presented as mean ± SEM. n = 4 or 5 repeated experiments with 6 biological replicates per group in each experiment. *p < 0.05 and ***p < 0.001 in two-way ANOVA analyses followed by Sidak tests. (B) Effects of SRC-2 on POMC-luciferase activity in fasted cells with or without FoxO1 knockdown. Data are presented as mean ± SEM. n = 4–7 repeated experiments with 6 biological replicates per group in each experiment. ***p < 0.001 in two-way ANOVA analyses followed by Sidak tests. (C) Hypothalamic mRNAs in fasted control and pomcSRC-2-KO mice. Data are presented as mean ± SEM. n = 14 or 15 mice per group. *p < 0.05 in t tests. (D) Interaction of endogenous SRC-2 and FoxO1 in the hypothalamus from chow- or HFD-fed male mice. (E) Body weight curves in male control and pomcSRC-2-KO littermates fed HFD ad libitum since 7 weeks of age. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05 in two-way ANOVA analyses repeated measurement followed by Sidak tests. (F) Fat or lean mass in 14-week-old male control and pomcSRC-2-KO littermates after 7 weeks HFD feeding. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05 in t tests. (G) Cumulative HFD intake in male control and pomcSRC-2-KO littermates fed HFD ad libitum since 7 weeks of age. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05 in two-way ANOVA analyses repeated measurement followed by Sidak tests. (H) Changes in body weight during a 3-week HFD feeding in mice receiving stereotaxic injections of AAV-FLEX-FoxO1 AAA or AAV-GFP into the ARH. Data are presented as mean ± SEM. n = 6–10 mice per group. * or #, p < 0.05 between POMC-Cre+ AAV-FLEX-FoxO1 AAA versus WT+ AAV-FLEX-FoxO1 AAA or versus pomcSRC-2-KO+AAV-FLEX-FoxO1 AAA in two-way ANOVA analyses repeated measurement followed by Sidak tests. (I and J) Changes in body weight (I) and fat mass (J) at the end of the 3-week HFD feeding in mice described in (H). Data are presented as mean ± SEM. n = 6–10 mice per group. *p < 0.05 in two-way ANOVA analyses followed by Sidak tests; p = 0.09 in t tests. (K) Cumulative HFD intake in mice described in (H). Data are presented as mean ± SEM. n = 6–10 mice per group. * or #, p < 0.05 between POMC-Cre+ AAV-FLEX-FoxO1 AAA versus WT+ AAV-FLEX-FoxO1 AAA or versus pomcSRC-2-KO+AAV-FLEX-FoxO1 AAA in two-way ANOVA analyses repeated measurement followed by Sidak tests.

Article Snippet: The proteins were analyzed by immunoblotting with rabbit monoclonal anti-FoxO1 antibody (1:3,000, #2880, Cell Signaling) or mouse monoclonal anti-FoxO1 antibody (1:3,000, #97635, Cell Signaling), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was rabbit anti-mouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Techniques: Luciferase, Activity Assay

Journal: Cell reports

Article Title: Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition

doi: 10.1016/j.celrep.2021.110075

Figure Lengend Snippet:

Article Snippet: The proteins were analyzed by immunoblotting with rabbit monoclonal anti-FoxO1 antibody (1:3,000, #2880, Cell Signaling) or mouse monoclonal anti-FoxO1 antibody (1:3,000, #97635, Cell Signaling), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was rabbit anti-mouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Transgenic Assay

Journal: Cell reports

Article Title: Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition

doi: 10.1016/j.celrep.2021.110075

Figure Lengend Snippet:

Article Snippet: The blot was probed with anti-Flag-HRP (1:3,000–10,000, A8592, Sigma), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Transgenic Assay, Control

Journal: British Journal of Pharmacology

Article Title: Haem oxygenase‐1 up‐regulation by rosiglitazone via ROS‐dependent Nrf2‐antioxidant response elements axis or PPARγ attenuates LPS‐mediated lung inflammation

doi: 10.1111/bph.14465

Figure Lengend Snippet: Rosiglitazone‐induced HO‐1 expression via NOX/ROS‐dependent c‐Src/Pyk2/Akt pathway. HPAEpiCs were (A) transfected with p47phox or NOX2 siRNA, and pretreated with APO or DPI, (B) c‐Srci II, (C) PF431396 or (D) Akti VIII for 1 h, and then incubated with vehicle or rosiglitazone (30 μM) for the indicated time intervals. Western blot was performed by using an anti‐phospho‐c‐Src, anti‐c‐Src, anti‐phospho‐Pyk2, anti‐Pyk2, anti‐phospho‐Akt or anti‐Akt antibody. Data are expressed as mean ± SEM, from five independent experiments (n = 5). # P < 0.05, significantly different from rosiglitazone alone.

Article Snippet: Anti‐β‐actin (C4) (mouse monoclonal antibody, Cat# sc‐47778 HRP, RRID:AB_2714189), anti‐c‐Src (SRC 2) (rabbit polyclonal antibody, Cat# sc‐18, RRID:AB_631324), anti‐Akt1/2/3 (H‐136) (rabbit monoclonal antibody, Cat# sc‐8312, RRID:AB_671714), anti‐Nrf2 (C‐20) (rabbit polyclonal antibody, Cat# sc‐722, RRID:AB_2108502), anti‐Keap1 (H‐190) (rabbit polyclonal antibody, Cat# sc‐33569, RRID:AB_2280949), anti‐Lamin A (H‐102) (rabbit polyclonal antibody, Cat# sc‐20680, RRID:AB_648148), anti‐ICAM‐1 (H‐108) (rabbit polyclonal antibody, Cat# sc‐7891, RRID:AB_647486) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Incubation, Western Blot