Fluorescently Tagged Podosome Proteins

Human myo1e and myo1f constructs tagged with EGFP have been previously described (Barger et al., 2019 (link)). mEmerald-Lifeact was a gift from Michael Davidson (Addgene #54148). Cofilin-EGFP was a gift from James Bamburg (Addgene #50859). Chicken regulatory light chain (RLC) tagged with EGFP was a gift from Klaus Hahn. WASP tagged with myc was a gift from Dianne Cox, and was subcloned into a pUB-Halo-C1 vector. CMV-GFP-NMHCII-A was a gift from Robert Adelstein (Addgene #11347). ARP3-mCherry (Addgene #27682) and mCherry-cortactin (Addgene #27676) were gifts from Christien Merrifield that were subcloned into EGFP-C1 and mEmerald-C1, respectively. Chicken paxillin was a gift from Chris Turner, which was subcloned into mScarlet-i-C1. Chicken vinculin was a gift from Kenneth Yamada (Addgene #50513) and subcloned into pUB-mEmerald-C1. Immunohistochemical staining to determine localization of select podosome-related proteins in relation to the actin teeth did not produce good results, which may be due to the adhesive and porous nature of the IgG-functionalized DAAMPs. As an alternative, we transfected the RAW macrophages with fluorescently tagged proteins. All transfections were accomplished by electroporation (Neon) using the manufacturer’s instructions.

Free full text: Click here

Vorselen D., Barger S.R., Wang Y., Cai W., Theriot J.A., Gauthier N.C, & Krendel M. (2021). Phagocytic ‘teeth’ and myosin-II ‘jaw’ power target constriction during phagocytosis. eLife, 10, e68627.

Publication 2021

mEmerald-Lifeact CMV-GFP-NMHCII-A ARP3-mCherry mCherry-cortactin

Actin Chicken Cofilin Cortactin Electroporation Gifts Human Light Macrophages Myo1f Neon Paxillin Podosome Proteins Teeth Transfections Vector Vinculin Wasp

Corresponding Organization :

Other organizations : Howard Hughes Medical Institute, University of Washington, SUNY Upstate Medical University, Stanford University, FIRC Institute of Molecular Oncology

Top 5 similar protocols

Variable analysis

independent variables

Myo1e and myo1f constructs tagged with EGFP
MEmerald-Lifeact
Cofilin-EGFP
Chicken regulatory light chain (RLC) tagged with EGFP
WASP tagged with myc
CMV-GFP-NMHCII-A
ARP3-mCherry
MCherry-cortactin
Chicken paxillin tagged with mScarlet-i
Chicken vinculin tagged with pUB-mEmerald-C1

dependent variables

Localization of select podosome-related proteins in relation to the actin teeth

control variables

RAW macrophages

positive controls

None specified

negative controls

None specified

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!