Collected cells were counted at collection point and lysed in 1 × Leammli buffer. Samples were separated on SDS-PAGE gels and transferred onto PVDF membranes (0.45 μm pore dimension). Membranes were blocked in 5% non-fat dry milk (VWR) for 1 h at room temperature. Subsequently, membranes were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used according to the manufacturers’ instructions: anti-AMPK (#5832, Cell Signaling Technology), phosphorylated AMPK (p-AMPK) (#8208, Cell Signaling Technology), BAF155 (#11956, Cell Signaling Technology), BAF170 (#12760, Cell Signaling Technology), BRG1 (#3538, Cell Signaling Technology), BRM (#11966, Cell Signaling Technology), Ezh2 (#5246, Cell Signaling Technology), INI1 (#8545, Cell Signaling Technology), PD-1 (#86163, Cell Signaling Technology), PD-L1 (#13684, Cell Signaling Technology), SUZ12 (#3737, Cell Signaling Technology), IL-6 (#12153, Cell Signaling Technology), and IL-1β (#12703, Cell Signaling Technology). The membranes were washed three times with TBS-T (0.01%) and incubated with HRP-conjugated secondary antibody (antirabbit IgG, #1705046, Bio-Rad) for 1 h in room temperature. After washing three times with TBS-T, the signal was detected using a chemiluminescent substrate (Western Bright Quantum, Advansta) and visualized using a UVTech gel blot imaging system.
Phosphorylated ampk p ampk
Manufactured by Cell Signaling Technology
Sourced in United States, China
Phosphorylated AMPK (p-AMPK) is a protein that has been post-translationally modified by the addition of a phosphate group. AMPK is an essential energy sensor that regulates cellular metabolism in response to changes in the AMP/ATP ratio. Phosphorylation of AMPK activates the enzyme, allowing it to play a key role in maintaining cellular energy homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (2 mentions)
Westernbright quantum, by Advansta (1 mentions)
Il 1β, by Cell Signaling Technology (1 mentions)
Suz12, by Cell Signaling Technology (1 mentions)
Baf155, by Cell Signaling Technology (1 mentions)
Baf170, by Cell Signaling Technology (1 mentions)
Anti ampk, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (1 mentions)
Pd l1, by Cell Signaling Technology (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Hypersensitive enhanced chemiluminescence kit, by Beyotime (1 mentions)
Gapdh, by Abways (1 mentions)
Pgc 1α, by Proteintech (1 mentions)
Protein lysis kit, by Beyotime (1 mentions)
Omni ecltm femto light chemiluminescence kit, by Ipsen (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sirtuin 1 sirt1, by Cell Signaling Technology (1 mentions)
Beyoecl moon kit, by Beyotime (1 mentions)
4 protocols using phosphorylated ampk p ampk
1
Western Blot Analysis of Epigenetic and Immune Regulators
2
Western Blot Analysis of Cell Signaling
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Total protein concentrations were examined using the Bicinchoninic Acid Protein Assay. Samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Skim milk was used for incubation with specific primary antibodies (1:1000 dilution) at 4° C overnight. The antibodies included phosphorylated AMPK (p-AMPK) (Cell Signaling Technology, Danvers, MA, United States), cyclin B1 (Diagbio, Hangzhou, China), Cdk1 (Abways, Beijing, China), p21 (Diagbio), C-myc (Diagbio), and GAPDH (Abways). The samples were incubated with secondary antibodies (1:2000 dilution) (Biosharp, Harjumaa, Estonia) at room temperature for 1 h. Protein bands were visualized using a hypersensitive enhanced chemiluminescence kit (Beyotime). The Bio-Rad gel imaging system was used to photograph gels, and ImageJ software was used for analysis.
3
Protein Expression Analysis of Hepatic AdipoR1/R2
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Protein extracts were extracted from liver tissues or HepG2 cells using the Protein Lysis Kit (Beyotime, China) and then separated on 12% SDS-PAGE gels. Then, gels were transferred to polyvinylidene difluoride (PVDF) membranes and PVDF membranes were incubated with primary antibodies against AdipoR1, AdipoR2 (Affinity Biosciences, China), AMPK, phosphorylated-AMPK (p-AMPK), LKB1, phospho-LKB1 (p-LKB1) (Cell Signaling Technology, MA, USA), SIRT1, nuclear factor erythroid-2-related factor 2 (Nrf2), carnitine palmitoyltransferase-1A (CPT1A), silent information regulator transcription 3 (SIRT3), PGC1α and GAPDH (Proteintech Group, USA) overnight at 4 °C. The PVDF membranes were washed and incubated with secondary antibodies at room temperature for 1h. Finally, Omni-ECLTM Femto Light Chemiluminescence Kit (EpiZyme, China) was used to detect specific protein expression. The images were obtained using Chemi-Lumin One Ultra (Tanon, China).
4
Western Blotting for Protein Analysis
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Cell or tissue was lysed using RIPA lysis buffer (Bmassay), and Western blot was carried out based on published literature (Li et al., 2019 (link)). In brief, protein extracts were separated in 10% SDS polyacrylamide gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking the membrane for 1 h, primary antibody was incubated overnight with gentle agitation. The primary antibodies for the target proteins included NPRA (Fabgennix), P21 (Proteintech), sirtuin 1 (SIRT1), PKG, AMPK and phosphorylated‐AMPK (p‐AMPK; Cell Signaling Technology). Subsequently, secondary antibody goat anti‐rabbit IgG or goat anti‐mouse IgG (ABclonal) was incubated at room temperature for 1 h. The blots were visualized employing a BeyoECL Moon kit (Beyotime). For probing multiple targets with the same membrane, stripping and re‐probing were performed. Briefly, the PVDF membrane was incubated with the stripping buffer containing 62.5 mM Tris–HCl (pH 6.8), 2% SDS and 100 mM β‐mercaptoethanol for 20 min at room temperature. After washing three times with TBST, the membrane was re‐incubated with primary and secondary antibodies.
The protein levels, normalized with GAPDH (Proteintech), were calculated by ImageJ (NIH).
The protein levels, normalized with GAPDH (Proteintech), were calculated by ImageJ (NIH).
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