Phosphorylated jak2

Manufactured by Cell Signaling Technology

Sourced in United States

Phosphorylated JAK2 is a lab equipment product that detects the phosphorylated form of the Janus Kinase 2 (JAK2) protein. JAK2 is a cytoplasmic tyrosine kinase involved in cell signaling pathways.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Phosphorylated JAK2 (pJAK2; (3 citations) Anti-phosphorylated JAK2 (2 citations)

5 protocols using phosphorylated jak2

Western Blot Analysis of Signaling Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

The proteins were extracted from the cell pellets. Proteins were boiled for 10 min and loaded onto 4–12% bis Tris gels (Invitrogen, Waltham, MA, USA) for separation. The separated proteins were then blotted onto polyvinylidene difluoride (PVDF) membranes (Invitrogen) with 20% (v/v) methanol in NuPage Transfer Buffer (Invitrogen) at 15 V for 4 h at 4°C. Tris-buffered saline containing 0.01% Tween 20 (TBST) in 5% skim milk (Difco, BD Biosciences, Oxford, UK) was used to block the membranes for 1 h. The blots were washed three times with TBST for 10 min and then incubated overnight at 4 °C with primary antibodies specific to the following target proteins: phosphorylated JAK1, phosphorylated JAK2, phosphorylated STAT1 (1:1000; Cell Signaling Technology, Cambridge, UK), CSF1, IL-6, PTPN6, RAC2, TNFα, IL-1α, IL-1β, phosphorylated STAT3, STAT3 ADORA2A, JAK1, JAK2, STAT1, SOCS3, Bax, Bcl-2, and β-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation, the blots were washed thrice with TBST and incubated for 1 h with a horse-radish peroxidase–conjugated secondary antibody (1:3000; Santa Cruz) at 25°C. Finally, the blots were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK).

Jo S., Baek A., Cho Y., Kim S.H., Baek D., Hwang J., Cho S.R, & Kim H.J. (2023). Therapeutic effects of polydeoxyribonucleotide in an in vitro neuronal model of ischemia/reperfusion injury. Scientific Reports, 13, 6004.

+ Open protocol

+ Expand

Immunoblotting for Stem Cell Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

For immunoblotting, cultured cells were harvested, washed with phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer as described previously (24 (link)). Antibodies used are listed as follows: rabbit antibodies against PRRX1 (Cat# ab211292) and Nanog (Cat# ab218524) were from Abcam (Shanghai, China). Rabbit antibodies against CD133 (Cat# 64326), SOX2 (Cat# 3579), OCT4 (Cat# 2750), phosphorylated-JAK2 (Cat# 3771), phosphorylation-STAT3 (Cat# 9145), and cleaved-PARP (Cat# 5625) were from Cell Signaling Technology (CST; Danvers, MA, USA). Rabbit antibodies against JAK2 (Cat# 17670-1-AP), mouse against IL-6 (Cat#66146-1-Ig), STAT3 (Cat# 60199-1-Ig), BCL2 (Cat# 60178-1-Ig), CNND1 (Cat# 60186-1-Ig), and PARP (Cat# 66520-1-Ig) and GAPDH (Cat# 60004-1-Ig) were from Proteintech (Rosemont, IL, USA). All the antibodies were used at 1:1,000 dilutions. The membranes, probed with the indicated primary antibodies, were subjected to the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit: Cat# 7074 and anti-mouse: Cat# 7076, CST, USA), and developed by enhanced chemiluminescence.

Zhong L., Tan W., Yang Q., Zou Z., Zhou R., Huang Y., Qiu Z., Zheng K, & Huang Z. (2022). PRRX1 promotes colorectal cancer stemness and chemoresistance via the JAK2/STAT3 axis by targeting IL-6. Journal of Gastrointestinal Oncology, 13(6), 2989-3008.

+ Open protocol

+ Expand

Colonic JAK2/STAT3 Signaling Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

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.

Ding P., Liu J., Li Q., Lu Q., Li J., Shi R., Shi L., Mao T., Ge D., Niu H., Peng G, & Wang Z. (2021). Investigation of the Active Ingredients and Mechanism of Hudi Enteric-Coated Capsules in DSS-Induced Ulcerative Colitis Mice Based on Network Pharmacology and Experimental Verification. Drug Design, Development and Therapy, 15, 4259-4273.

+ Open protocol

+ Expand

Western Blot Analysis of Key Signaling Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols

The primary antibodies are PD-L1 (CST #13684, Cell Signaling Technology), PD-L1 (17952- 1-AP, Santa Cruz), STAT3 (CST #9139, Cell Signaling Technology), phosphorylated STAT3 (CST #9145, Cell Signaling Technology), JAK2 (17670-1-AP, Proteintech), phosphorylated JAK2 (CST #4406, Cell Signaling Technology), AKT (10176-2-AP, Proteintech), phosphorylated AKT (66444-1-Ig, Proteintech), ERK (CST #4696, Cell Signaling Technology), phosphorylated ERK (CST #3510, Cell Signaling Technology), PARP-1 (sc-8007, Santa Cruz), and GAPDH (60004-1-Ig, Proteintech). Goat anti-rabbit antibody and mouse anti-rabbit antibody conjugated to HRP were purchased from Biosharp. all antibodies and reagents were stored and used according to the manufacturer’s instructions. Briefly, tissues were homogenized, mixed with 5X loading buffer and boiled until denaturation. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes are sealed with 5% skim milk and incubated overnight at 4°C with the primary antibody. The membranes were then washed, incubated with secondary antibodies for 1 hour at room temperature, and visualized with SuperSignal West Pico plus chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA).

Wang Y., Zheng K., Xiong H., Huang Y., Chen X., Zhou Y., Qin W., Su J., Chen R., Qiu H., Yuan X., Wang Y, & Zou Y. (2021). PARP Inhibitor Upregulates PD-L1 Expression and Provides a New Combination Therapy in Pancreatic Cancer. Frontiers in Immunology, 12, 762989.

+ Open protocol

+ Expand

Apoptosis Induction Assay Protocols

Check if the same lab product or an alternative is used in the 5 most similar protocols

Hematoxylin and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma. Arsenic trioxide for injection was purchased from Double Heron Pharmaceutical Co., LTD. Cryptotanshinone was purchased from Chengdu Must, Bio-technology Co., LTD. Antibodies against cleaved-caspase-3, cleaved-caspase-9, cleaved poly(ADP-ribose) polymerase, Bax, Bak, XIAP, Mcl-1, Bcl-2, Bcl-xl, survivin, phosphorylated-JAK2, and phosphorylated-STAT3^Tyr705 were purchased from Cell Signaling Technology, while β-actin antibody was purchased from Sigma-Aldrich. An Annexin V/PI binding kit was purchased from Santa Cruz Biotechnology, Inc. RIPA Lysis Buffer and a BCA Protein Assay Kit were purchased from Beyotime. Immobilon ECL was purchased from Millipore. Rhodamin-labeled goat anti-mouse immunoglobulin G (IgG) and DAPI were obtained from Hangzhou Dawei Biotech Co., LTD.

Shen L., Zhang G., Lou Z., Xu G, & Zhang G. (2017). Cryptotanshinone enhances the effect of Arsenic trioxide in treating liver cancer cell by inducing apoptosis through downregulating phosphorylated- STAT3 in vitro and in vivo. BMC Complementary and Alternative Medicine, 17, 106.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!