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: Acta Biochimica et Biophysica Sinica

Article Title: Saikosaponin D reverses epinephrine- and norepinephrine-induced gemcitabine resistance in intrahepatic cholangiocarcinoma by downregulating ADRB2/glycolysis signaling

doi: 10.3724/abbs.2023040

Figure Lengend Snippet: Effect of ADRB2 on the proliferation and gemcitabine resistance of iCCA cells (A) Western blot analysis was used to analyze the expression level of ADRB proteins in RBE cells and HuCCT1 cell lines. (B) The efficiency of ADRB2 knockout was determined by real-time quantitative PCR and western blot analysis (***P<0.001 vs NTC). (C) Effects of ADRB2 knockout on NE-stimulated iCCA cell proliferation by CCK8 assay and are presented as a growth curve (*** P<0.001 vs norepinephrine+NTC). (D) Effects of ADRB2 knockout on NE-induced gemcitabine resistance in iCCA cells. Data are presented as the mean±SD (n=3).

Article Snippet: Primary antibodies against ADRB2 (#8513), ABCG2 (#42078), MRP1 (#72202), HK2 (#2867), and GLUT1 (#73015) were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Western Blot, Expressing, Knock-Out, Real-time Polymerase Chain Reaction, CCK-8 Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Saikosaponin D reverses epinephrine- and norepinephrine-induced gemcitabine resistance in intrahepatic cholangiocarcinoma by downregulating ADRB2/glycolysis signaling

doi: 10.3724/abbs.2023040

Figure Lengend Snippet: Effects of ADRB2 on aerobic glycolysis and ABC transporter levels in iCCA cells (A,B) Measurement of the extracellular acidification rate (ECAR) in iCCA cells influenced by NE, E and ICI118551 with the XFe24 Extracellular Flux Analyser. (C) Glycolytic variations (glycolysis, glycolytic capacity and glycolytic reserve) in each group were summarized from raw data (**P<0.01, ***P<0.001 vs control; #P<0.05 vs NE). (D) Glucose consumption in each group was detected using a glucose assay kit (***P<0.001 vs control; ###P<0.001 vs NE). (E) Production of lactic acid in each group was assayed using a Lactic acid production detection kit (***P<0.001 vs control; ##P<0.01 vs NE). (F) Western blot analysis was used to analyse the protein expression levels of HK2, GLUT1, ABCG2 and MRP1in RBE cells and HuCCT1 cell lines from each group. Data are presented as the mean±SD (n=3).

Article Snippet: Primary antibodies against ADRB2 (#8513), ABCG2 (#42078), MRP1 (#72202), HK2 (#2867), and GLUT1 (#73015) were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Control, Glucose Assay, Western Blot, Expressing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Saikosaponin D reverses epinephrine- and norepinephrine-induced gemcitabine resistance in intrahepatic cholangiocarcinoma by downregulating ADRB2/glycolysis signaling

doi: 10.3724/abbs.2023040

Figure Lengend Snippet: Effect of SSD on ADRB2 expression in iCCA cells stimulated with NE The effects of SSD on the levels of ADRB2 were determined by western blot analysis (A) and real-time quantitative PCR (B). Data are presented as the mean±SD (n=3). ***P<0.001 vs NE.

Article Snippet: Primary antibodies against ADRB2 (#8513), ABCG2 (#42078), MRP1 (#72202), HK2 (#2867), and GLUT1 (#73015) were purchased from Cell Signaling Technology (Danvers, USA).

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

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Saikosaponin D reverses epinephrine- and norepinephrine-induced gemcitabine resistance in intrahepatic cholangiocarcinoma by downregulating ADRB2/glycolysis signaling

doi: 10.3724/abbs.2023040

Figure Lengend Snippet: Effect of SSD on ADRB2/glycolysis signaling in iCCA cells stimulated with NE (A) Measurement of the extracellular acidification rate (ECAR) in iCCA cells influenced by NE with/without SSD (2.5 and 5 μM) with the XFe24 Extracellular Flux Analyzer. (B) Glycolytic variations (glycolysis, glycolytic capacity and glycolytic reserve) in each group were summarized from raw data (*P<0.05, **P<0.01, ***P<0.001 vs NE). (C) Glucose consumption in each group was detected using a glucose assay kit (**P<0.01, ***P<0.001 vs NE). (D) Production of lactic acid in each group was assayed using a Lactic acid production detection kit (**P<0.01, ***P<0.001 vs NE). (E) Western blot analysis was used to analyse the protein expression levels of HK2, GLUT1, ABCG2 and MRP1 in RBE cells and HuCCT1 cell lines from each group. Data are presented as the mean±SD ( n=3).

Article Snippet: Primary antibodies against ADRB2 (#8513), ABCG2 (#42078), MRP1 (#72202), HK2 (#2867), and GLUT1 (#73015) were purchased from Cell Signaling Technology (Danvers, USA).

Techniques: Glucose Assay, Western Blot, Expressing

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