Abstract The foot domains of polymerases from the Paramyxoviridae family mediate attachment of the viral polymerases to their nucleocapsid templates during viral RNA synthesis. Detailed structural and biophysical characterization of these foot domains reveals that they share a common structure, but when not bound to the nucleocapsid they may exist as intrinsically unstructured chains, molten globules, or highly stable folded domains. With hundreds of paramyxovirus sequences now known, the diverse family of foot domains provides a valuable system to investigate the physical and evolutionary processes that dictate protein stability. To enable rapid and robust evaluation of the relative stability of these domains, we tested the applicability of the split β‐lactamase assay. This assay couples β‐lactamase activity to the folding of a protein inserted into a loop on the enzyme, with antibiotic resistance being conferred in proportion to the stability of the insert. In this work, five foot domains of broadly varying stability were tested with this assay. We constructed a bespoke vector to facilitate the use of this assay, and we introduced a novel statistical approach that generates a probability‐based ranking of relative stability from the experimental data. We find that this assay reproduces the order of stabilities obtained through biophysical approaches. Though classical definitions of stability, evaluated using traditional biophysical techniques, are ill‐defined for proteins of low intrinsic stability, this work finds that the split β‐lactamase assay can be fruitfully adapted in such cases, and can be made more convenient and interpreted more rigorously through the innovations we describe. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Preparation of screening vector suBLA for ligation‐independent cloning Basic Protocol 2 : Ligation‐independent cloning of inserts into screening vector suBLA Basic Protocol 3 : Use of PCR to check for inserts Basic Protocol 4 : Dilution series assay Basic Protocol 5 : Scoring of dilution assay Basic Protocol 6 : Statistical analysis of dilution assay
Abstract This protocol describes a method for endogenous protein tagging in postmitotic primary neurons using homology‐independent targeted integration (HITI), a CRISPR/Cas9‐based approach that exploits non‐homologous end joining (NHEJ). Unlike homology‐directed repair (HDR), HITI does not require cell division, making it uniquely suited for postmitotic neurons. SpCas9 is delivered by a replication‐incompetent simian immunodeficiency lentiviral vector (pLenti SIV), while the sgRNA, donor tag cassette, and a fluorescent plasma membrane marker are co‐packaged in a single AAV vector. The two vectors are co‐delivered at DIV5, and endogenous tagging is assessed at DIV15–23. The workflow is illustrated using Junctophilin‐3 (JPH3) tagged with mEmerald at its endogenous locus. Airyscan microscopy imaging of endogenously tagged JPH3 at native expression levels revealed its localization at endoplasmic reticulum–plasma membrane (ER–PM) contact sites and uncovered a periodic distribution of ER–PM junctions along the dendritic arbor, enabling identification of a previously unrecognized ladder‐like organization of the ER in neuronal dendrites. Genomic integration of the tag is validated at both 5' and 3' junctions by next‐generation sequencing. The protocol can be adapted to other neuronal proteins by redesigning the guide RNA and donor sequence, and supports fluorescent proteins as well as epitope and self‐labeling tags. The plasma membrane marker can also be replaced with other fluorescent reporters or markers for intracellular organelles to assess infection efficiency and verify subcellular localization of the protein of interest. The full workflow takes 5–6 weeks. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Protocols included in this article: Basic Protocol 1 : sgRNA design and HITI donor vector cloning Basic Protocol 2 : Primary rat neuron culture and viral co‐transduction Basic Protocol 3 : Live‐cell imaging of endogenously tagged proteins Support Protocol 1 : Production of lentiviral and AAV particles Support Protocol 2 : Validation of knock‐in by next‐generation sequencing (NGS)
Abstract Functional enrichment analysis is a cornerstone of bioinformatics data analysis, enabling researchers to put high‐throughput transcriptomics or proteomics data into biological context by identifying relevant pathways, processes, and mechanisms. The clusterProfiler R package is widely used for its robust statistical methods and versatile visualization options, including bar plots, dot plots, and enrichment maps. Whereas enrichment maps reveal relationships between terms through shared gene content, their static R implementations provide limited interactive exploration and customization for complex networks with hundreds of enriched terms and also provide limited built‐in support for displaying node‐level properties such as pathway coverage or expression directionality. We present a complete workflow for enrichment analysis and network visualization. The protocols cover performing over‐representation analysis and gene set enrichment analysis using clusterProfiler, calculating term similarity between enriched terms, and exporting networks to Cytoscape using our cyemapplot function. The approach demonstrates three complementary visualization styles: basic networks reveal functional modules and term relationships, pie chart networks quantify pathway coverage to prioritize complete versus partial perturbations, and differentially expressed gene networks integrate expression directionality and regulatory heterogeneity within biological processes. By revealing shared gene content, expression patterns, and network topology, this approach helps researchers move beyond independent term lists to extract richer mechanistic insights from high‐throughput datasets. The support protocols cover established clusterProfiler and enrichplot functionality required to generate enrichment results, whereas the Basic Protocol describes the main methodological contribution of this work, the cyemapplot function and its three visualization styles. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol : Advanced network visualization using Cytoscape Support Protocol 1 : Installing required software and packages Support Protocol 2 : Preparing gene expression data Support Protocol 3 : Performing enrichment analysis and calculating term similarity
Abstract Proteolytic cleavage is a fundamental biological process that regulates protein maturation, signaling pathways, immune responses, and disease progression. Accurate identification of protease substrate cleavage sites is critical for understanding protease function and substrate specificity. However, experimental determination of cleavage sites remains labor‐intensive and time‐consuming, motivating the development of efficient and accurate computational prediction methods. Here, we present a detailed protocol for using ProsperousPlus, a computational platform designed for large‐scale prediction of protease‐specific substrate cleavage sites and construction of customizable models for these predictions. ProsperousPlus integrates multiple sequence‐derived scoring functions with an advanced machine learning framework that automates the construction of optimized predictive models. The framework's workflow includes three major functional modules: (1) prediction of cleavage sites using pre‐trained models covering more than 100 proteases, (2) training of custom protease‐specific models using user‐provided datasets, and (3) deployment of trained models for large‐scale prediction tasks. This protocol describes the required hardware and software environment, dataset preparation strategies, model training procedures, and interpretation of prediction results, including probability scores and multiple evaluation metrics such as accuracy and AUC. In addition, model interpretation using Shapley additive explanations (SHAP) enables identification of influential sequence features contributing to cleavage specificity. ProsperousPlus can be used either through a standalone software for large‐scale, more frequent analyses or through a user‐friendly web server for small‐scale, ad hoc use. This protocol provides a practical and reproducible workflow for researchers seeking to analyze protease substrate specificity, construct customized prediction models, and perform high‐throughput proteolytic analysis. © 2026 Wiley Periodicals LLC. Basic Protocol : Predicting protease‐specific substrate cleavage sites and constructing and deploying protease‐specific models using the standalone software version of ProsperousPlus Alternate Protocol : Using the web server version
Abstract Tissue stiffness changes markedly in many pathological conditions, such as cancer and fibrosis, influencing cellular behavior and function. To investigate these effects in vitro , it is necessary to culture cells on substrates whose mechanical properties can be tuned to mimic both normal and diseased tissue environments. This protocol provides a detailed, reproducible procedure for preparing polyacrylamide (PAA) hydrogels with defined stiffness values that are functionalized with extracellular matrix (ECM) proteins and suitable for cell culture and imaging studies. It outlines the steps for cleaning and functionalizing glass coverslips, preparing acrylamide and bis‐acrylamide mixtures of desired concentrations, initiating polymerization, and conjugating ECM proteins to support cell adhesion. The protocol further describes the process of seeding cells onto the hydrogels, followed by live‐cell imaging, chemical fixation, and staining for visualization of cellular structures and protein localization. Practical notes and troubleshooting tips are also included for maintenance of hydrogel integrity and optimization of experimental reproducibility. The workflow enables systematic investigation of how substrate stiffness regulates cell morphology, migration, cytoskeletal organization, and other mechanosensitive responses. Overall, this protocol provides a reliable framework for preparing and using tunable PAA‐based hydrogels in studies of cell–matrix interactions, mechanotransduction, and disease‐related changes in tissue mechanics. It is adaptable to a wide range of cell types and compatible with various imaging modalities, offering a versatile platform for mechanobiology research. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Preparation of polyacrylamide‐based hydrogels of tunable rigidities and functionalization for cell culture Support Protocol 1 : Preparation of reagents for hydrogel preparation Basic Protocol 2 : Staining for live‐cell imaging and visualization of cellular structures and protein localization Basic Protocol 3 : Chemical fixation and immunofluorescence staining for visualization of cellular structures and protein localization Support Protocol 2 : Preparation of reagents and materials for cell fixation and immunostaining.
