Biochemistry

Post-translational modifications play important roles in controlling protein function and can lead to altered protein stability. Protein stability can be determined after treatment with the protein synthesis inhibitor Cycloheximide. Cycloheximide is a translational inhibitor that inhibits protein synthesis via cytoplasmic ribosomes. Here we describe how to measure the stability of MYC2 in the context of regulation by FERONIA receptor kinase. First, we describe how to measure MYC2 stability in wild-type and feronia mutant; then we describe similar assays in transgenic plants expressing MYC2-FLAG and MYC2^A12-FLAG (12 FERONIA phosphorylation sites are mutated to Alanine and the mutant protein is stabilized). MYC2 can be induced by mechanical touch, which can be a confounding factor in protein level measurement. In this protocol, we take that into consideration and try to achieve more accurate measurement.

In health, the high-speed airflow associated with cough represents a vital backup mechanism for clearing accumulated mucus from our airways. However, alterations in the mucus layer in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) leads to the mucus layer adhered to the airway surfaces, representing the nidus of chronic lung infection. To understand what is different about diseased mucus and why cough clearance is defective, there is a need for techniques to quantify the strength of the interactions limiting the ability of airflow to strip mucus from the airway surface (i.e., adhesive strength) or tear mucus apart (i.e., cohesive strength). To overcome the issues with measuring these properties in a soft (i.e., low elastic modulus) mucus layer, we present here novel peel-testing technologies capable of quantifying the mucus adhesive strength on cultured airway cells and cohesive strength of excised mucus samples. While this protocol focuses on measurements of airway mucus, this approach can easily be adapted to measuring adhesive/cohesive properties of other soft biological materials.

Structural stability of the capsid core is a critical parameter for the productive infection of a cell by a retrovirus. Compromised stability can lead to premature core disassembly, exposure of replication intermediates to cytosolic nucleic acid sensors that can trigger innate antiviral responses, and failure to integrate the proviral genome into the host DNA. Thus, core stability is a critical feature of viral replicative fitness. While there are several well-described techniques to assess viral capsid core stability, most are generally time and labor intensive. Recently, our group compared the relative stability of murine leukemia virus capsid cores using an in vitro detergent-based approach combined with ultracentrifugation against the popular fate of capsid assay. We found that both methods reached similar conclusions, albeit the first method was a significantly simpler and faster way to assess relative capsid core stability when comparing viral mutants exhibiting differences in core stability.