polyclonal rabbit anti-phospho-acc  (Cell Signaling Technology Inc)

Journal: Pharmacological research

Article Title: Alternative splicing of Mff regulates AMPK-mediated phosphorylation, mitochondrial fission and antiviral response.

doi: 10.1016/j.phrs.2024.107414

Figure Lengend Snippet: Fig. 2. Multiple assays for AMPK-mediated Mff phosphorylation in MEFs in response to mitochondrial energetics. (a–c) Conventional SDS-PAGE and immunoblot analysis of Mff and AMPK phosphorylation in WT MEFs. The indicated letters (a, b, c) correspond to the endogenous Mff bands observed in WT MEFs. (a) WT MEFs were treated with increasing concentrations of oligomycin (10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM) for 30 min. Mff band shift (band c to b) was detected by anti-Mff antibodies. Mff KO MEFs were used as negative control. (b) WT MEFs were treated with oligomycin (1 and 0.3 μM) for 30 min. Phosphorylation of endogenous Mff was detected using an anti-phospho-Mff (S146) antibody. (c) WT MEFs were treated with 20 μM CCCP for the indicated times and harvested with or without washout. The activation of AMPK signaling was confirmed by phosphorylation of AMPKα at Thr172 or ACC at Ser79 using specific antibodies. (d–f) Conventional SDS-PAGE or Phos-tag SDS-PAGE and immunoblot analysis of Mff in Mff KO MEFs stably expressing FLAG-tagged Mff isoform 4. Black and white arrowheads indicate phospho-Mff and unphospho-Mff in SDS-PAGE or AMPK-phospho-Mff and AMPK-unphospho-Mff in Phos-tag SDS-PAGE, respectively. (d) Mff KO MEFs stably expressing FLAG-tagged Mff isoform 4 were treated with 20 μM CCCP and harvested with or without washout. *Non-specific band. (e, f) Phos-tag SDS-PAGE analysis of Mff in response to AMPK activity. Mff KO MEFs stably expressing FLAG-tagged Mff isoform 4 WT (e, f) or S146A mutant (f) were treated with 2 mM AICAR or 20 μM compound C (Comp. C) for 2 h. The response of Mff to AMPK signaling was analyzed by Phos-tag SDS-PAGE. Mff KO MEFs were used as negative control. *Non-specific band.

Article Snippet: Antibodies used in this study: rabbit polyclonal anti-Mff (1:1000, Proteintech, 17090–1-AP), mouse monoclonal anti-Drp1 (1:250 for IF, BD Biosciences, 611113), mouse monoclonal anti-β-Actin (1:10000, Sigma, A2228), mouse monoclonal anti-FLAG M2 (1:1000, Sigma, #F1804), rabbit polyclonal anti- phospho-Mff (Ser146) (1:1000, Cell Signaling, #49281), rabbit monoclonal anti-phospho-AMPKα (Thr172) (1:1000, Cell Signaling, #2535), rabbit polyclonal anti-AMPKα (1:1000, Cell Signaling, #2532), rabbit polyclonal anti-phospho-ACC (Ser79) (1:1000, Cell Signaling, #3661).

Techniques: Phospho-proteomics, SDS Page, Western Blot, Electrophoretic Mobility Shift Assay, Negative Control, Activation Assay, Stable Transfection, Expressing, Activity Assay, Mutagenesis

Journal: Frontiers in Endocrinology

Article Title: Hepatic steatosis induced by nicotine plus Coca-Cola™ is prevented by nicotinamide riboside (NR)

doi: 10.3389/fendo.2024.1282231

Figure Lengend Snippet: Nic plus Coke administration increases the lipogenesis marker in the liver. (A) Representative western blot analysis of SREBP1 cleavage (SREBP1c). β-Actin levels are shown as a loading control. Molecular weight markers are depicted in KDa. (B) Quantification of the western blot of SREBP1c. (C) Representative western blot analysis of p-ACC. β-Actin levels are shown as a loading control. Molecular weight markers are depicted in KDa. (D) Quantification of the western blot of p-ACC. Protein levels of SREBP1c and p-ACC were normalized to β-Actin and expressed as the mean ± S.E.M. (the fold of change relative to the control). n= 5 - 6 per group. Statistical difference is indicated by * compared to Sal+Water vs Nic+Coke (P< 0.05).

Article Snippet: We then incubated the membranes with the following first antibodies (diluted in blocking solution) including, rabbit polyclonal phospho-Acetyl-CoA Carboxylase (p-ACC) (1:1000) (3661; Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal phospho-AMP-activated protein kinase (p-AMPK) (1:1000) (2535; Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal HO-1 (1:2000) (AB13243; Abcam, San Francisco, CA, USA), mouse monoclonal Mitochondrial complex cocktail (1:3000) (AB110413; Abcam, San Francisco, CA, USA), rabbit polyclonal NAMPT (1:250) (AB45890; Abcam, San Francisco, CA, USA), rabbit polyclonal PGC1 α (1:1000) (AB54481; Abcam, San Francisco, CA, USA), rabbit polyclonal Sirt1 (1:2000) (AB12193; Abcam, San Francisco, CA, USA), rabbit polyclonal SOD2 (1:500) (sc-30080; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal SREBP1 (1:500) (AB3259; Abcam, San Francisco, CA, USA), and rabbit polyclonal β -Actin (1:4000) (AB8227; Abcam, San Francisco, CA, USA). β -Actin was used as a loading control, all antibodies were incubated overnight at 4°C with constant shaking.

Techniques: Marker, Western Blot, Control, Molecular Weight

Journal: iScience

Article Title: Exercise-acclimated microbiota improves skeletal muscle metabolism via circulating bile acid deconjugation

doi: 10.1016/j.isci.2023.106251

Figure Lengend Snippet: Exercise-acclimated microbiota treatment improves glucose tolerance in HFD-fed mice (AN) Body and tissue weights (n = 8, A–C), blood chemistry (n = 8, D and E), blood glucose concentrations during oral glucose tolerance tests (n = 8, GTT) (F) and insulin tolerance tests (ITT) (n = 8, G), glycogen content (n = 7–8, H), mRNA levels of PGC1α and COX4-1 (n = 8, I and J), AMPKα Thr172 (n = 7, K) and ACC Ser212 (n = 6–7, L) phosphorylation, membrane content of GLUT4 (n = 7, M), and COX activity (n = 8, N) in gastrocnemius muscle in recipient mice fed chow or HFD for 8 weeks. Phosphorylation levels were correlated with total content of each target in immunoblotting (K–M). The solid line shows absolute values, and the dotted line shows relative values in the ITT (G). RS; recipient from sedentary donor, RT; recipient from trained donor. ∗p < 0.05, and ∗∗p < 0.01 between groups. Results are presented as the mean ± SE.

Article Snippet: Rabbit polyclonal anti-phospho ACC (Ser212) , Cell Signaling Technology , Cat#3661S; RRID: AB_330337.

Techniques: Phospho-proteomics, Membrane, Activity Assay, Western Blot

Journal: iScience

Article Title: Exercise-acclimated microbiota improves skeletal muscle metabolism via circulating bile acid deconjugation

doi: 10.1016/j.isci.2023.106251

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho ACC (Ser212) , Cell Signaling Technology , Cat#3661S; RRID: AB_330337.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Protein Extraction, Gene Expression, Software, Mass Spectrometry, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: The transcription factors TFEB and TFE3 link the FLCN-AMPK signaling axis to innate immune response and pathogen resistance

doi: 10.1016/j.celrep.2019.02.102

Figure Lengend Snippet: Key Resource Table

Article Snippet: Rabbit polyclonal p-ACC (S79) , Cell Signaling , Cat#3661.

Techniques: Virus, Plasmid Preparation, shRNA, Clone Assay, Recombinant, Cell Viability Assay, Gene Expression, Microarray, Over Expression, Software