Immunoblotting of JAK2 and STAT Signaling

Immunoblotting was performed as previously described (20 (link)). For examination of the protein involved in pro-survival signaling pathway, cell lysis from empty-, JAK2-, or JAK2^V617F-expressing macrophages were extracted and subpackaged equally for three groups (each group included empty, JAK2 and JAK2^V617F lysates), followed by separation on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After semi-dry transfer, the membranes were sequentially probed with the indicated antibodies. Anti-p-JAK2 (T1007/1008) (#3776), anti-JAK2 (D2E12) (#3230), anti-p-STAT1 (T701) (#7649), anti-STAT1 (D1K9Y) (#14994), anti-p-STAT3 (Tyr705, D3A7) (#9145), anti-STAT3 (D3Z2G) (#12640), anti-p-STAT5 (Tyr694, C11C5) (#9359), anti-STAT5 (D206Y) -(#94205), anti-p-p38 (Thr180/Tyr182, D3F9) (#4511), anti-p38 (D13E1) (#8690), anti-p-AKT (Ser473, D9E) (#10019), anti-AKT (#9272), anti-p-JNK (Thr183/Tyr185, 81E11) (#4668), and anti-JNK (#9252) antibodies were purchased from Cell Signaling Technology. Anti-HK1 (19662-1-AP), anti-HK2 (22029-1-AP), anti-PKM1 (15821-1-AP), anti-flag (Sigma), and anti-β-actin (66009-1-Ig) antibodies were purchased from ProteinTech.

Free full text: Click here

Li R., Sun N., Chen X., Li X., Zhao J., Cheng W., Hua H., Fukatsu M., Mori H., Takahashi H., Ohkawara H., Fukami M., Okamoto M., Hamazaki Y., Zheng K., Yang J, & Ikezoe T. (2021). JAK2V617F Mutation Promoted IL-6 Production and Glycolysis via Mediating PKM1 Stabilization in Macrophages. Frontiers in Immunology, 11, 589048.

Publication 2020

anti-JAK2 (D2E12) anti-STAT1 (D1K9Y) anti-p38 (D13E1) anti-JNK anti-β-actin (66009-1-Ig)

Actin Antibodies Cell Jak2 Macrophages Membranes Protein Sds page Signaling pathway Stat1 Stat3 Stat5

Corresponding Organization : Fukushima Medical University

Top 5 similar protocols

Variable analysis

independent variables

Empty-
JAK2-
JAK2^V617F^-expressing macrophages

dependent variables

Protein levels of p-JAK2, JAK2, p-STAT1, STAT1, p-STAT3, STAT3, p-STAT5, STAT5, p-p38, p38, p-AKT, AKT, p-JNK, JNK, HK1, HK2, PKM1

control variables

Protein load (cell lysates were extracted and subpackaged equally for the three groups)

controls

No positive or negative controls were explicitly mentioned in the input protocol.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

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!