Protein samples were harvested from liver tissues. After blocking with 5% non-fat milk, the PVDF membranes containing resolved proteins were incubated overnight at 4°C using rabbit anti-mouse antibodies against AMPK (1:1000, Abcam), phosphorylated HDAC4 (1:1000, Abcam), p-AMPK (1:1000, Santa Cruz), HDAC4 (1:1000, Santa Cruz), G6Pase (1:1000, Abcam) and α-microtubule protein (1:1000, Santa Cruz). The membranes were then incubated with peroxidase-conjugated secondary antibodies (Sigma-Aldrich) for 1 h and developed using an ECL system acquired from GE Healthcare Life Science. Relative expression was calculated using NIH-Image J1.51p 22 (NIH).
G6pase
Manufactured by Abcam
Sourced in United States, United Kingdom
G6Pase is an enzyme that catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. It plays a key role in gluconeogenesis and glycogenolysis.
Automatically generated - may contain errors
Lab products found in correlation
Pepck, by Abcam (4 mentions)
Streptozotocin (stz), by Merck Group (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
P ampk, by Santa Cruz Biotechnology (1 mentions)
Hdac4, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Agarose, by Merck Group (1 mentions)
Foxo1, by Abcam (1 mentions)
P pi3k, by Abcam (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Sglt2, by Abcam (1 mentions)
Hrp conjugated secondary antibody against rabbit igg, by Agilent Technologies (1 mentions)
Hnf4a, by Santa Cruz Biotechnology (1 mentions)
Chrebp, by Abcam (1 mentions)
Sr b1, by Santa Cruz Biotechnology (1 mentions)
Hmgcr, by Santa Cruz Biotechnology (1 mentions)
Srebp1, by Santa Cruz Biotechnology (1 mentions)
12 protocols using g6pase
1
Western Blot Analysis of AMPK and HDAC4
2
Renal Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As described previously [16 (link)], kidneys were homogenized in a fivefold volume of 20 mmol/L Tris buffer with proteinase inhibitors. After centrifugation at 5,000g for 15 min, the supernatants were centrifuged at 48,000g for 60 min at 4 °C to obtain the cytosolic fraction and membrane fraction. The 50 µg of renal proteins were applied to a 4–20% gradient gel and electroblotted onto polyvinylidene fluoride membranes. The specific protein bands were identified using rabbit polyclonal antibodies for sodium glucose co-transporter 2 (SGLT2) (Abcam, Tokyo, Japan), G6Pase (Abcam), or PEPCK (Abcam) at 1:500 dilution followed by a horseradish peroxidase-conjugated secondary antibody against rabbit immunoglobulin G (Dako, Glostrup, Denmark) and the bands were visualized with diaminobenzidine reaction. The band stained with anti-beta-actin antibody (Abcam) was used as a loading control. The density of the bands was analyzed using the National Institutes of Health Image software program (version 1.63).
3
Herbal Formulation for Diabetes Management
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMix (15 g dendrobium, 20 g astragalus, 8 g schisandra, 15 g pueraria, 15 g salvia, 15 g rehmannia and 8 g earthworms) was purchased from Guoyitang Clinic, Fujian University of TCM. Metformin (glucophage) tablets were purchased from the Sino-American Shanghai Squibb Pharmaceutical Co., Ltd. (Shanghai, China), with a production specification of 0.85 g/tablet (national medicine approval number H20023370). The other experimental reagents included an insulin ELISA detection kit (cat no. F6403; Westang Biotechnology Co., Ltd., Shanghai, China), a blood glucose meter and blood glucose strips (Yuyue Medical Equipment & Supply Co., Ltd. (Nanjing, China), TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), anhydrous ethanol and isopropyl alcohol. Agarose was purchased from Sigma-Aldrich (Merck, KGaA, Darmstadt, Germany). The following antibodies were used: Insulin receptor (InsR, cat. no. 3025), Akt (cat. no. 4685) and phosphorylated (p)-Akt (cat. no. 9611) (all Cell Signaling Technology, Inc., Danvers, MA, USA), anti-β-actin (cat. no. SAB1305567; Sigma-Aldrich; Merck KGaA), PI3K, p-PI3K, FoxO1, PEPCK and G6pase (all Abcam, Cambridge, MA, USA). A SYBR-Green I quantitative PCR kit (Takara Bio, Inc., Otsu, Japan) was purchased from Thermo Fisher Scientific, Inc. and PCR primers were produced by Invitrogen (Thermo Fisher Scientific, Inc.).
4
Immunohistochemical Analysis of Metabolic Enzymes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue specimens embedded in wax were processed for immunohistochemistry, as previously described.23 (link) Sections (2 μm in thickness) were dewaxed and incubated with citrate buffer (pH 6) at 100°C for 20 minutes in order to retrieve the antigens and then sections were incubated with 3% H2O2 and blocking serum followed by polyclonal antibodies against SGLT2 (Abcam), G6Pase (Abcam), or PEPCK (Abcam) at 1:200 dilution. The sections were subsequently incubated with an HRP-conjugated secondary antibody against rabbit IgG (Dako Denmark A/S) at 1:50 dilution, and HRP labeling was detected using DAB reaction. The sections were then counterstained with periodic acid-Schiff staining before being examined under a light microscope. Two sections from each animal were stained at the same time with concurrent processing, and evaluated using the unlabeled blind procedure.
5
Antibody Sourcing for Lipid Metabolism
6
Evaluation of SI and Pioglitazone on Diabetes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SI was obtained from a local company in Thailand; its composition is shown in Table 1 . Pioglitazone hydrochloride (PZ) was acquired from Berlin Pharmaceutical Industry Co., Ltd.. Primary antibodies, including insulin receptor-β (IR-β), IRS-1, phosphoenolpyruvate carboxykinase-1 (PEPCK/PCK-1), p-Akt (Ser 473), and Akt, and a secondary antibody were procured from Cell Signaling Technology, Inc.. Primary antibody against glucose 6-phosphatase (G-6-Pase) was obtained from Abcam. STZ and all chemicals were purchased from Sigma-Aldrich Co. and Merck Millipore.
7
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue proteins were extracted using 300 µL of radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1.0% sodium deoxycholate, 0.1% SDS) in the presence of a cocktail of protease inhibitor (Roche Diagnostics, Basel, Switzerland)) and phosphatases (Roche Diagnostics, Basel, Switzerland)) and frozen at −80 °C. Protein concentration was determined using BCA Protein assay kit Protein (Sigma, St. Louis, MO, USA) with a standard curve of bovine serum albumin. A total of 30 µg of each sample was separated by electrophoresis in polyacrylamide gels (Tris-glycine SDS-polyacrylamide 10%) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (0.45 µm). The primary antibodies and their dilution were FKBP5 1:1000 (12210S, Cell Signaling), G6Pase 1:500 (83690, Abcam, Cambridge, United Kingdom), and GAPDH 1:5000 (MAB374, Sigma). GAPDH was used as a loading control. The images were scanned using C-Digit and quantified using Image Studio Digits (LICOR Bioscience, Lincon, NE, USA).
8
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
20 μg of total protein was separated on NuPAGE 4–12% BIS-TRIS Gels (Invitrogen, USA) and transferred onto a PVDF membrane. Membrane was blocked in blocking buffer (Sigma, USA) for 1 hr. The immunoblotting was performed at 4°C overnight with shaking using antibodies against p-ERK (Cell Signaling, USA), p-IR, IR, PEPCK, G-6-Pase (Abcam, USA), ERK, p-PPARγ, PPARγ, and GAPDH (Santa Cruz, USA). After washing, the membrane was incubated in a 1 : 5000 dilution of a secondary antibody (goat anti-rabbit IgG, Thermo Scientific, USA) at room temperature for 1 hr. Protein bands were visualized using ECL kit (Thermo Scientific, USA).
9
Immunoblotting analysis of mitochondrial and metabolic proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected Hepa 1–6 cells were lysed or mice liver tissues were homogenised in RIPA lysis buffer containing protease and phosphatase inhibitors. Lysates (20–40 μg) were separated using SDS-PAGE (6 %–12 %), transferred on nitrocellulose or PVDF membranes and blocked with 5 % BSA followed by incubation overnight at 4 °C with primary antibodies against GRP75 (Cat no. 2799), FACL4 (Cat no. 155282), MFN1 (Cat no. 57602), MFN2 (Cat no. 56889), SIGMA1R (Cat no. 53852), VDAC1 (Cat no. 14734), G6Pase (Cat no. 83690), p-IRS1(S307) (Cat no. 5599), MCU (Cat no. 121499): Abcam, Cambridge, UK; IP3R1/2/3 (Cat no. 85685), PCK1 (Cat no. 12740), FBP1 (Cat no. 59172), p-JNK1/2 (Cat no. 9255), ERK1/2 (Cat no. 4695), p-ERK1/2 (Cat no. 4377), p38 (Cat no. 9228), p-P38 (Cat no. 9216), IRS1 (Cat no. 2390), p-AKT (Ser473, Cat no. 4060) and AKT (Cat no. 4685): Cell Signaling Technology, MA, USA; JNK (Cat no. sc7435), HSC 70 (Cat no. sc7298), β-actin (Cat No. sc81178), vinculin (Cat no. sc73614): SantaCruz Biotechnology, Texas, USA); Pcx (SAB2500845, Sigma, St. Louis, USA) and PACS2 (PA5-72866, Thermo, MA, USA) using specific antibodies. Immunoreactive bands were detected using ECL Chemiluminescence kit (Gbiosciences, USA) vinculin, β-actin or HSC70 were used as loading controls.
10
Molecular Mechanisms of Hepatic Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
STZ was purchased from Sigma (SaintLouis, Missouri, USA). RNAiso Plus was purchased from TaKaRa (Dalian, China). Rabbit polyclonal SIK1 antibody was purchased from Novus Biologicals, LLC (Cat #: 82417, Littleton, USA). Rabbit polyclonal SREBP-1c antibody, G6Pase and CRTC2 (S171) antibody were purchased from Abcam (Cat #: ab28481, Cat #: ab83690 and Cat #: ab203187, Cambrige, UK). Rabbit polyclonal CRTC2 antibody, SIK1 (S577) antibody, SIK1 (T182) antibody, FAS antibody and ACC antibody were purchased from Proteintech Group, Inc. (Cat #:12497-1-AP, Cat #: S4530-2, Cat #: S4529-2, Cat #:10624-2-AP and Cat #:21923-1-AP, Rosemont, USA). Rabbit monoclonal PEPCK antibody and β-actin antibody were purchased from Cell Signaling Technology, Inc. (Cat #: 12940 and Cat #: 4967, Danvers, Massachusetts, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG was purchased from Bioworld Technology, Inc. (Minnesota, USA) as secondary antibody.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!