Expression and Membrane Targeting of β1-integrin-ECFP in Mammalian Cells

β1-integrin is a transmembrane glycoprotein that heterodimerizes with multiple α subunits and functions as a key receptor for extracellular matrix proteins. It contains a large extracellular ligand-binding domain, a single-pass transmembrane region, and a short cytoplasmic tail involved in intracellular signaling that supports cell adhesion, migration, and tissue development. Fluorescent β1-integrin fusion proteins are challenging to image in standard cell lines because endogenous β1-integrin competes with the fusion protein for maturation and α-subunit pairing, often resulting in aggregation or endoplasmic reticulum retention, and the consequent perinuclear fluorescence. This article describes a straightforward transient expression strategy that yields properly processed β1-integrin-ECFP at the plasma membrane in murine HC11 cells. The method uses a bicistronic construct that expresses β1-integrin-ECFP while simultaneously reducing endogenous Itgb1 via a short hairpin RNA targeting its 3' untranslated region, thereby sparing the recombinant transcript. This approach enables robust membrane localization of the fusion protein and enrichment at structures consistent with focal adhesions. © 2026 Wiley Periodicals LLC. Basic Protocol: Construction and transient expression of a bicistronic β1-integrin-ECFP/shItgb1 plasmid to achieve plasma membrane targeting in HC11 cells Support Protocol 1: Glass coverslip cleaning and surface functionalization for cell adhesion Support Protocol 2: Cell fixation using paraformaldehyde.