The rabbit polyclonal PIPKIγ antibody was generated and purified as described previously (6 (link)). Antibodies against phosphorylated AKT (pSer473), total AKT, phosphorylated ERK1/2 (pThr202/Tyr204), total ERK1/2, phosphorylated STAT3 (pTyr705), total STAT3, phosphorylated JAK2 (pTyr1007/1008) and total JAK2 were procured from Cell Signaling Technology (Danvers, MA, USA). Antibody for β-actin was purchased from Sigma-Aldrich.
Total jak2
Manufactured by Cell Signaling Technology
Sourced in United States
Total JAK2 is a lab equipment product that measures the total amount of JAK2 protein in a sample. JAK2 is a protein that plays a crucial role in various cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Total stat3, by Cell Signaling Technology (5 mentions)
Phospho jak2, by Cell Signaling Technology (4 mentions)
Antibodies against α sma and β actin, by Merck Group (2 mentions)
Recombinant human tgf β1, by R&D Systems (2 mentions)
Fugene hd transfection reagent, by Promega (2 mentions)
E cadherin, by BD (2 mentions)
P jak2 y1007 1008, by Cell Signaling Technology (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Antibody for β actin, by Merck Group (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
Phospho hsl ser563, by Cell Signaling Technology (1 mentions)
Total stat5, by Cell Signaling Technology (1 mentions)
Hif 1α, by Merck Group (1 mentions)
Hif 2α, by R&D Systems (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Boster Bio (1 mentions)
Phosphorylated stat3, by Cell Signaling Technology (1 mentions)
Phosphorylated jak2, by Cell Signaling Technology (1 mentions)
11 protocols using total jak2
1
Antibody Generation and Purification
2
Recombinant TGF-β1 Pathway Regulation
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Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). Fugene HD transfection reagent was from Promega (Madison, WI, USA). Antibodies against PGC-1α and fibronectin were from Santa Cruz (Dallas, TX, USA) or Abcam (Cambridge, MA, USA). Antibodies against phospho-Stat3 (Tyr705), Total-Stat3, Total-Smad2/Smad3, Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425), TGF-β, Total-Jak2, and Phospho-Jak2 were all from Cell Signaling Technology (Danvers, MA, USA). E-cadherin was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Antibodies against α-SMA and β-actin were from Sigma-Aldrich (St. Louis, MO, USA). The selective antibiotics, zeocin was purchased from Invitrogen (Carlsbad, CA, USA).
3
TGF-β1 Signaling Pathway Analysis
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Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Fugene HD transfection reagent was from Promega (Madison, WI). Antibodies against phospho-Stat3 (Tyr705), Total-Stat3, Total-Smad2/Smad3, Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425), TGF-β, Total-Jak2, and Phospho-Jak2 were all from Cell Signaling Technology (Danvers, MA). Fibronectin was from Santa Cruz (Dallas, Texas). E-cadherin was purchased from BD Biosciences (Franklin Lakes, NJ). Antibodies against α-SMA and β-actin were from Sigma-Aldrich (St. Louis, MO). Specific antibodies against Prdx1, Prdx2, Prdx3, Prdx4, Prdx5, and Prdx6 were gifts from Dr. Ho Zoon Chae (Chonnam National University, Korea).
4
Western Blot Analysis of Cell Signaling
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Tissue or cells were lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor and phosphatase inhibitor cocktails. Western blots were probed with the following antibodies: total JAK2, total STAT5, phospho-JAK2 (Tyr1007/1008) phospho-STAT5 (Tyr694) , ATGL , HSL and phospho-HSL (Ser563) purchased from Cell Signaling Technology (Danvers, MA, USA); EPOR (R&D Systems, Minneapolis, MN, USA); HIF-1α (Sigma-Aldrich, Saint Louis, MO, USA); HIF-2α (R&D Systems) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
5
Colonic JAK2/STAT3 Signaling Analysis
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The protein expressions of total JAK2, phosphorylated JAK2, STAT3, and phosphorylated STAT3 (Cell Signaling Technology, U.S.A) in colonic tissues were analyzed using Western blotting. Proteins in colon tissues of four mice in each group were extracted using the RIPA lysis buffer (Boster, Wuhan, China) and were quantified using an Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). The proteins (20 μg from each sample) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. After subjection to blocking with 5% bovine serum albumin for 1 h, the membrane was incubated with primary antibodies (1:5,000) at 4 °C overnight. After conducting three washes with Tris-buffered saline containing 0.1% Tween-20, the membrane was incubated with secondary antibodies (1:5,000) at room temperature for 2 h. Finally, The GelDoc Go gel imaging system is used for exposure and development, and the Image J image analysis system is used to analyze the grayscale optical density of the target band. (Bio-Rad Company, America). All protein levels were normalized to those of β-actin.
6
Thyroid Gland Protein Analysis
7
Signaling Pathway Profiling in GH-Stimulated Cells
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Phospho-JAK2 and total JAK2 were obtained from Cell Signaling Technology (USA). Phospho-STAT5 and total STAT5 were obtained from Santa Cruz (USA). Antiphosphotyrosine antibody (clone 4G10) was purchased from Upstate Biotechnology. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse antibodies and recombinant GH were purchased from Sigma (Sigma–Aldrich Company, St. Louis, MO, USA). 125I-GH was prepared using chloramine T according to published procedures, and the specific activity of GH was 70 to 130 μCi/μg protein. Lipofectamine Plus and pCDNA3.1 were obtained from Invitrogen (Invitrogen, Carlsbad, CA, USA). The ImmunoPure Fab Preparation kit, Cell Lysis Buffer, IP kit, Enhanced chemiluminescence (ECL) and the BCA kit were purchased from pierce (Pierce Biotechnology Inc, Rockford, IL, USA). Porcine GH receptor cDNAs were obtained from Takara Biotechnology Co., Ltd (Dalian, China). The pre-stained molecular weight standards and all tissue reagents were obtained from Gibco (Gibco, Grand Island, NY, USA). The porcine IGF-1 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Hua Yi Medical and Biological Laboratories Co., Ltd (Changchun, China). Unless stated otherwise, all other reagents were from Sigma-Aldrich (USA).
8
Protein Expression Analysis in HK-2 Cells and Mice Kidneys
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Total proteins of cultured HK-2 cells and kidneys of the mice were extracted with cell lysis buffer supplemented with protease and phosphatase inhibitor cocktails. The concentration of proteins was detected with a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. After being blocked with 5% skim milk for 1 h, the membranes were incubated overnight at 4°C with antibodies directed against total STAT3 and phospho-STAT3-Tyr705 (1:2,000, Cell Signaling Technology), total JAK2 (1:1,000, Cell Signaling Technology), phospho-JAK2-Tyr1007/1008 (1:2,000, Abcam), cleaved caspase-3 (1:1,000, Cell Signaling Technology), Bax (1:2,000, ProteinTech), Bcl-2(1:1,000, ProteinTech), gasdermin D (GSDMD, 1:1,000, Abcam), and caspase-1 (1:2,000, Huabio). The next day, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence substrate and quantified by ImageJ software ver. 1.8.
9
Western Blot Analysis of Cellular Signaling
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Protein lysates were extracted, quantified, resolved and transferred onto a PVDF membrane (Millipore) and probed with primary and secondary antibodies prior to detection using chemiluminescence system (GE Healthcare). The following antibodies were used: ADAR1 (1:1000, Cell Signaling Technology, #14175), ADAR1 p150 specific (1:1000, abcam, ab168809), ADAR2 (1:1000, Sigma-Aldrich, SAB1405426), SCD1 (1:1000, abcam, ab19862), ATF4 (1:1000, Santa Cruz Biotechnology, sc-390063), CHOP (1:500, Cell Signaling Technology, #2895), p-eIF2α (1:1000, Cell Signaling Technology, #9721), total eIF2α (1:1000, Cell Signaling Technology, #9722), p-STAT3 (1:1000, Cell Signaling Technology, #9134), total STAT3 (1:1000, Cell Signaling Technology, #9139), p-JAK2 (1:1000, Cell Signaling Technology, #3771), total JAK2 (1:1000, Cell Signaling Technology, #3230), LRP5 (1:1000, abcam, ab36121), LRP6 (1:1000, abcam, ab134146), β-catenin (1:1000, BD Biosciences, #610153), AXIN2 (1:1000, abcam, ab109307), cyclin D1 (1:1000, Santa Cruz Biotechnology, sc-753), KHDRBS1 (1:1000, abcam, ab86239), β-actin (1:5000, Sigma-Aldrich, A5316), Histone H3 (1:1000, abcam, ab24834), α-Tubulin (1:1000, Sigma-Aldrich, T9026). Images were captured using BioRad ImageLab Touch software (version 2.4.0.03).
10
Western Blot Analysis of Signaling Proteins
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Total protein was collected from the cells after various treatments. For Western blots, a previously described procedure was applied [28 (link)]. The following primary antibodies were used: p-Jak2 (Y1007/1008), total Jak2, p-Stat3 (Y705), p-Stat3 (S727), and total Stat3 were purchased from Cell Signaling Technology. Antibodies for gp130, gp80, Bmi-1, Oct4 and GAPDH were from Santa Cruz. Mcl-1 and CyclinD1 antibodies were obtained from Abcam. The secondary anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz. All the experiments were performed at least twice and quantification data with statistical analysis was summarized in Figure S19 .
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