rabbit anti total ampk α antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION

doi: 10.1101/2023.05.05.539524

Figure Lengend Snippet: (A) Graph showing the mitochondrial OCR of C2C12 cells overexpressing pMIG GFP and pMIG RBM3-GFP, basal OCR and OCR after treatment with oligomycin (1 uM), FCCP (3 uM), antimycin and rotenone (1.5 uM), where the x-axis represents time in minutes and the y-axis represents oxygen consumption rate in pMol/min. (B) Bar graph measuring the basal respiration, maximum respiration (OCR after FCCP addition), spare respiratory capacity (basal respiration-maximum respiration) and ATP-linked respiration (basal respiration-respiration after oligomycin addition) of C2C12 cells overexpressing pMIG-GFP and pMIG-RBM3 GFP where the y-axis represents oxygen consumption rate in pMol/Min (n=2). (C) Heat map showing levels of TCA metabolites using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (D) Heat map showing levels of TCA metabolites using media (48 hrs.) from C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (E) Graphical representation of levels of acetyl-CoA using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). mRNA expression levels of glycolytic genes (F) PKM1 , (G) PKM2 in C2C12 overexpressing pMIG-GFP control and pMIG-RBM3. (H) Western blot analysis of glycolytic protein levels (PKM1, PKM2) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (I) mRNA expression levels of glycolytic genes PKM1 , PKM2 in mouse primary myoblasts overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (J) Western blot analysis of AMPK-beta and 4E-BP1 using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (K) Western blot analysis of acetyl-CoA carboxylase (ACC) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (L) Bar graph quantifying phosphorylated/total 4E-BP1, ACC and AMPK-beta respectively. *, **, *** represents p-value < 0.05, 0.01 and 0.001 respectively.

Article Snippet: Primary antibodies MyHC (Invitrogen 14650382), MF-20 (DHSB AB_2147781) MYOG (Invitrogen MA5-11486), MyoD1 (Santa Cruz SC-377460), RBM3 (Invitrogen PA5-51976), beta-ACTIN (CST 4967S), beta-Tubulin (CST 2146), PKM1(CST D30G6), beta-Actin (CST 4967) PKM2 (CST D78A4), PDH (CST 3205), SDHA (CST 5839), phospho-4E-BP1 (CST 2855), total 4E-BP1 (CST 9452), phospho-AMPK-alpha (CST 2535), total-AMPK-alpha (CST 5831), phospho-AMPK-beta (CST 4186), total-AMPK-beta (CST 4150), phospho-ACC (CST 11818) and total ACC (CST 3676) were used in 1:1000 dilution and incubated at 4 0 C overnight.

Techniques: Control, Expressing, Western Blot

Journal: bioRxiv

Article Title: PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION

doi: 10.1101/2023.05.05.539524

Figure Lengend Snippet: (A) Graph showing the mitochondrial OCR of C2C12 cells overexpressing pMIG GFP and pMIG RBM3-GFP, basal OCR and OCR after treatment with oligomycin (1 uM), FCCP (3 uM), antimycin and rotenone (1.5 uM), where the x-axis represents time in minutes and the y-axis represents oxygen consumption rate in pMol/min. (B) Bar graph measuring the basal respiration, maximum respiration (OCR after FCCP addition), spare respiratory capacity (basal respiration-maximum respiration) and ATP-linked respiration (basal respiration-respiration after oligomycin addition) of C2C12 cells overexpressing pMIG-GFP and pMIG-RBM3 GFP where the y-axis represents oxygen consumption rate in pMol/Min (n=2). (C) Heat map showing levels of TCA metabolites using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (D) Heat map showing levels of TCA metabolites using media (48 hrs.) from C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (E) Graphical representation of levels of acetyl-CoA using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). mRNA expression levels of glycolytic genes (F) PKM1 , (G) PKM2 in C2C12 overexpressing pMIG-GFP control and pMIG-RBM3. (H) Western blot analysis of glycolytic protein levels (PKM1, PKM2) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (I) mRNA expression levels of glycolytic genes PKM1 , PKM2 in mouse primary myoblasts overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (J) Western blot analysis of AMPK-beta and 4E-BP1 using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (K) Western blot analysis of acetyl-CoA carboxylase (ACC) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (L) Bar graph quantifying phosphorylated/total 4E-BP1, ACC and AMPK-beta respectively. *, **, *** represents p-value < 0.05, 0.01 and 0.001 respectively.

Article Snippet: Primary antibodies MyHC (Invitrogen 14650382), MF-20 (DHSB AB_2147781) MYOG (Invitrogen MA5-11486), MyoD1 (Santa Cruz SC-377460), RBM3 (Invitrogen PA5-51976), beta-ACTIN (CST 4967S), beta-Tubulin (CST 2146), PKM1(CST D30G6), beta-Actin (CST 4967) PKM2 (CST D78A4), PDH (CST 3205), SDHA (CST 5839), phospho-4E-BP1 (CST 2855), total 4E-BP1 (CST 9452), phospho-AMPK-alpha (CST 2535), total-AMPK-alpha (CST 5831), phospho-AMPK-beta (CST 4186), total-AMPK-beta (CST 4150), phospho-ACC (CST 11818) and total ACC (CST 3676) were used in 1:1000 dilution and incubated at 4 0 C overnight.

Techniques: Control, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Red ginseng extract improves skeletal muscle energy metabolism and mitochondrial function in chronic fatigue mice

doi: 10.3389/fphar.2022.1077249

Figure Lengend Snippet: RGE activates AMPK/PGC-1α cascade in TA muscle of multiple stress factors-induced chronic fatigue mice. (A) . Representative western blotting images of p-AMPKα, t-AMPKα, PGC1α and GAPDH. (B,C) . Relative expression of p-AMPKα, t-AMPKα and PGC1α were quantified. n = 3 independent blots. (D) . Analysis of mtDNA/nuclear DNA by qPCR, the relative expression of NADH dehydrogenase 1 (mtDNA) to TFAM (nuclear DNA) was quantified using real-time quantitative PCR to determine the relative copy number differences of mtDNA. n = 6 per group. Mice from control or chronic fatigue syndrome model group were treated with water or different dose of RGE (200, 400 or 600 mg/kg). Compared with control group, * p < 0.05, ** p < 0.01, *** p < 0.001; compared with CFS group, # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: After blocking with 5% bovine serum albumin (BSA) for 2 h, the membranes were incubated with the following primary antibodies overnight at 4°C: rabbit monoclonal to phospho-AMPK (1:500; catalog 2535, Cell Signaling Technology, United States), total-AMPK (1:500; catalog 2603, CST), ACO2 (1:1000; catalog 6571T, CST), rabbit polyclonal to PGC1α (1:800; catalog ab54481, Abcam, United States), mouse monoclonal to NDUFB8 (1:1000; catalog 459210, Abcam), GAPDH (1:2000; catalog 97166, CST).

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