Journal: The FASEB Journal

Article Title: α-Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through PHD3/ADRB2 pathway

doi: 10.1096/fj.201700670R

Figure Lengend Snippet: PCR primer sequences and amplification parameters

Article Snippet: C2C12 cells were ncubated overnight in rabbit anti-PHD3 (bs-0532R; Bioss Antibodies) and rabbit anti-ADRB2 (bs-0947R; Bioss Antibodies) at 4°C.

Techniques: Amplification, Sequencing

Journal: The FASEB Journal

Article Title: α-Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through PHD3/ADRB2 pathway

doi: 10.1096/fj.201700670R

Figure Lengend Snippet: ADRB2 is target of PHD3 in response to AKG. A) C2C12 cells were induced differentiation for 4 d, and anti-PHD3 was used to pull down PHD3 binding protein. Precipitated samples were used for SDS-PAGE, and then strips were stained by Coomassie Brilliant Blue. B) C2C12 myotubes were treated by 2 mM AKG for 48 h, and anti-PHD3 and anti-ADRB2 were used to precipitate PHD3 and ADRB2, respectively. Precipitated samples was subjected to immunoblotting of ADRB2 or PHD3 (n = 3 group). C) C2C12 myotube samples from control or 2 mM AKG–treated group were pulled down by anti-ubiquitin antibody and subjected to immunoblotting of ADRB2 (n = 3/group). D) Immunoblots and quantification of ADRB2 in normal or PHD3-overexpressing C2C12 myotubes treated with 2 mM AKG for 48 h (n = 6/group). E, F) Representative images (E) and quantification (F) of ADRB2 in C2C12 myotubes treated with 0 or 2 mM AKG for 48 h (n = 6/group). G) Immunoblots and quantification of ADRB2 in C2C12 myotubes treated with 0 or 2 mM AKG for 48 h (n = 6/group). H) cAMP levels in C2C12 myotubes treated with 0 or 2 mM AKG for 48 h (n = 6/group). I–K) Immunoblots (I) and quantification (J, K) of p-PKA (J), PKA, p-CREB (K), and CREB (K) in C2C12 myotubes treated with 0 or 2 mM AKG for 48 h (n = 6/group). Data are presented as means ± sem and were analyzed by 1-way ANOVA followed by post hoc Bonferroni tests for panel D, and nonpaired Student’s t test for all others. β-Actin served as housekeeping gene. *P < 0.05 compared to control.

Article Snippet: C2C12 cells were ncubated overnight in rabbit anti-PHD3 (bs-0532R; Bioss Antibodies) and rabbit anti-ADRB2 (bs-0947R; Bioss Antibodies) at 4°C.

Techniques: Binding Assay, SDS Page, Staining, Western Blot

Journal: The FASEB Journal

Article Title: α-Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through PHD3/ADRB2 pathway

doi: 10.1096/fj.201700670R

Figure Lengend Snippet: Pharmacologic inhibition of ADRB2 blocked inhibitory effects of AKG on skeletal muscle atrophy and protein degradation. A–C) Immunoblots (A) and quantification (B, C) of p-PKA and PKA (B) and p-CREB and CREB (C) in C2C12 myotubes treated with vehicle, 2 mM AKG, 10 μM ICI, or 2 mM AKG + 10 μM ICI for 48 h (n = 6/group). D, E) Representative images (D) and quantification (E) of long-life protein Click-It AHA in C2C12 myotubes treated with vehicle, 2 mM AKG, 10 μM ICI, or 2 mM AKG + 10 μM ICI for 48 h (n = 6/group). F, G) Representative images (F) and quantification (G) of fiber diameter of C2C12 myotubes treated with vehicle, 2 mM AKG, 10 μM ICI, or 2 mM AKG + 10 μM ICI for 48 h (n = 6/group). H–K) Immunoblots (H) and quantification (I–K) of pFoxO1 and FoxO1 (I), MuRF1 (J), and MAFbx (K) in C2C12 myotubes treated with vehicle, 2 mM AKG, 10 μM ICI, or 2 mM AKG + 10 μM ICI for 48 h (n = 6/group). L, M) Representative images (L) and quantification (M) of propidium iodide (PI, red)-positive MuRF1 (green) cells (yellow nucleus indicated by white arrows) in gastrocnemius muscle from male C57BL/6J mice 3 h after intraperitoneal injection with vehicle, 1 g/kg AKG, 5 μg/kg ICI, or 1 g/kg AKG + 5 μg/kg ICI (n = 6). N) 3-MeHis in gastrocnemius muscle from male C57BL/6J mice 3 h after intraperitoneal injection with vehicle, 1 g/kg AKG, 5 μg/kg ICI, or 1 g/kg AKG + 5 μg/kg ICI (n = 6). O–R) Immunoblots (O) and quantification (P–R) of pFoxO1 and FoxO1 (P), MuRF1 (Q), and MAFbx (R) in gastrocnemius muscle from male C57BL/6J mice 3 h after injected intraperitoneally with vehicle, 1 g/kg AKG, 5 μg/kg ICI, or 1 g/kg AKG + 5 μg/kg ICI (n = 6). Data are presented as means ± sem and were analyzed by 1-way ANOVA, followed by post hoc Bonferroni tests. β-Actin served as housekeeping gene. *P < 0.05 compared to control.

Article Snippet: C2C12 cells were ncubated overnight in rabbit anti-PHD3 (bs-0532R; Bioss Antibodies) and rabbit anti-ADRB2 (bs-0947R; Bioss Antibodies) at 4°C.

Techniques: Inhibition, Western Blot, Injection

Journal: The FASEB Journal

Article Title: α-Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through PHD3/ADRB2 pathway

doi: 10.1096/fj.201700670R

Figure Lengend Snippet: ADRB2 knockdown abolished inhibitory effects of AKG on muscle atrophy and protein degradation. A) mRNA expression of ADRB2 in gastrocnemius muscle from male C57BL/6J mice injected intramuscularly with LV-shScrambled or LV-shADRB2. B–D) Immunoblots (B) and quantification (C, D) of p-PKA and PKA (C) and p-CREB and CREB (D) in gastrocnemius muscle from male C57BL/6J mice receiving LV-shScrambled, LV-shScrambled + AKG, LV-shADRB2, or LV-shADRB2 +AKG. E–J) Gastrocnemius weight (E), gastrocnemius muscle fiber size (F, G), muscle grip (H), high-speed running time (I), and slow-speed running time (J) of male C57BL/6J mice receiving LV-shScrambled, LV-shScrambled + AKG, LV-shADRB2, or LV-shADRB2 + AKG. K–N) Immunoblots (K) and quantification (L–N) of pFoxO1 and FoxO1 (L), MuRF1 (M), and MAFbx (N) in gastrocnemius muscle from male C57BL/6J mice receiving LV-shScrambled, LV-shScrambled + AKG, LV-shADRB2, or LV-shADRB2 + AKG. O, P) Representative images (O) and quantification (P) of propidium iodide (PI, red) positive MuRF1 (green) cells (yellow nucleus indicated by white arrows) in gastrocnemius muscle from male C57BL/6J mice receiving LV-shScrambled, LV-shScrambled + AKG, LV-shADRB2, or LV-shADRB2 + AKG. Q) Levels of 3-MeHis in gastrocnemius muscle from male C57BL/6J mice receiving LV-shScrambled, LV-shScrambled + AKG, LV-shADRB2, or LV-shADRB2 + AKG. Data are presented as means ± sem and were analyzed by nonpaired Student’s t test for panel A, and 1-way ANOVA, followed by post hoc Bonferroni tests for all others. β-Actin served as housekeeping gene. *P < 0.05 compared to control.

Article Snippet: C2C12 cells were ncubated overnight in rabbit anti-PHD3 (bs-0532R; Bioss Antibodies) and rabbit anti-ADRB2 (bs-0947R; Bioss Antibodies) at 4°C.

Techniques: Expressing, Injection, Western Blot

Journal:

Article Title: Different properties of P2X 7 receptor in hippocampal and cortical astrocytes

doi: 10.1007/s11302-009-9137-3

Figure Lengend Snippet: P2X7 receptor activation induces IL-1β release from cortical but not from hippocampal astrocytes. a Western blot analysis for IL-1β on cell lysates and supernatants of cortical (AC) and hippocampal cells (AH), primed 6 h with 100 ng/ml LPS and incubated with 100 μM BzATP for 30 min. Note the presence of the cytokine only in the supernatant of cortical astrocytes. b IL-1β detection by ELISA in the supernatants collected from hippocampal and cortical astrocytes primed 6 h with 100 ng/ml LPS and exposed to BzATP for 30 min. Values are presented as mean ± SE picograms per milliliter and are normalized to protein concentration of cell extracts. c Quantitative analysis of active caspase-1 by FLICA assay. Note the increase in the number of active caspase-1 upon BzATP exposure in cortical but not hippocampal astrocytes. d ELISA evaluation of IL-1β levels in the supernatants of cortical astrocytes maintained in static condition or mechanically stimulated with or without apyrase (one-way ANOVA, post hoc Dunn’s method, p = 0.003, n = 3) or the P2X7 antagonist oATP, showing that astrocyte-derived ATP induces IL-1β release from cortical cultures

Article Snippet: Mouse Abs against GFAP, Abs against P2X 7 (C-term and N-term), and IL-1β were from Alomone Labs (Israel).

Techniques: Activation Assay, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Protein Concentration, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: The Sympathetic Nervous System Contributes to the Establishment of Pre-Metastatic Pulmonary Microenvironments

doi: 10.3390/ijms231810652

Figure Lengend Snippet: Neuro-immune cell interactions in pre-metastatic lungs. ( A ) Interactions between sympathetic nerve fibers and non-endothelial cells. An NF-L-positive neuron interacted with a non-endothelial cell in pre-metastatic lungs. Enlarged image is the section enclosed by the broken line. Scale Bar: 10 µm. ( B ) Neuro-immune cell interaction. Pre-metastatic lungs were stained with anti-CD11b and anti-Th antibodies and observed using a confocal microscope. A Th-positive neuron interacted with CD11b-positivel cells. Scale Bar: 10 µm. ( C ) Co-culture of neurons and macrophages in vitro. Differentiated PC12 cells were co-cultured with J774.1 cells. Neuro-immune cell interactions were also found in vitro. Scale Bar: 10 µm. ( D ) Induced cytokine expression by the β2 adrenaline receptor agonist. J774.1 cells were stimulated with Terbutaline, a β2 adrenaline receptor selective agonist, or CL316,243, a β3 adrenaline receptor selective agonist; mRNA expression levels of S100a8 were tested by qPCR ( n = 3, * p < 0.05). ( E ) β2 adrenaline receptor-mediated up-regulation of S100a8 . Expression of S100a8 was up-regulated by TERB and abolished by pre-incubation with ICI118,551, a β2 selective antagonist ( n = 3, * p < 0.05). ( F – H ) Adrb2 expression in MDSCs. Pre-metastatic lungs were stained with anti-Adrb2, anti-CD11b, anti-CD11c, and anti-F4/80 antibodies and analyzed using a flow cytometer. Adrb2-positive cells were divided into CD11b + F4/80 + and CD11b − CD11c + populations. Both groups expressed Adrb2, and Adrb2 expression was induced in bone marrow-derived macrophages defined as CD11b + F4/80 + , but not in alveolar macrophages defined as CD11b − CD11c + ( n = 5, * p < 0.05).

Article Snippet: Alexa647-conjugated anti-Adrb2 antibody was purchased from Bioss (Cat. No. BS-0947R-A647, Woburn, MI, USA).

Techniques: Staining, Microscopy, Co-Culture Assay, In Vitro, Cell Culture, Expressing, Incubation, Flow Cytometry, Derivative Assay