The selective degradation of nuclear components via autophagy, termed nucleophagy, is an essential process observed from yeasts to mammals and crucial for maintaining nucleus homeostasis and regulating nucleus functions. In the budding yeast Saccharomyces cerevisiae, nucleophagy occurs in two different manners: one involves autophagosome formation for the sequestration and vacuolar transport of nucleus-derived vesicles (NDVs), and the other proceeds with the invagination of the vacuolar membrane for the uptake of NDVs into the vacuole, termed macronucleophagy and micronucleophagy, respectively. This chapter describes methods to analyze and quantify activities of these nucleophagy pathways in yeast.

Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.

Connexins are essential gap junction proteins that play pivotal roles in intercellular communication in various organs of mammals. Connexin-43 (Cx43) is expressed in various components of the immune system, and there is extensive evidence of its participation in inflammation responses. The involvement of Cx43 in macrophage functionality involves the purinergic signaling pathway. Macrophages contribute to defenses against inflammatory reactions such as bacterial sepsis and peritonitis. Several assays can identify the presence and activity of Cx43 in macrophages. Real-time polymerase chain reaction (PCR) can measure the relative mRNA expression of Cx43, whereas western blotting can detect protein expression levels. Using immunofluorescence assays, it is possible to analyze the expression and observe the localization of Cx43 in cells or tissues. Moreover, connexin-mediated gap junction intercellular communication can be evaluated using functional assays such as microinjection of fluorescent dyes or scrape loading-dye transfer. The use of selective inhibitors contributes to this understanding and reinforces the role of connexins in various processes. Here, we discuss these methods to evaluate Cx43 and macrophage gap junctions.

GelBox is open-source software that was developed with the goal of enhancing rigor, reproducibility, and transparency when analyzing gels and immunoblots. It combines image adjustments (cropping, rotation, brightness, and contrast), background correction, and band-fitting in a single application. Users can also associate each lane in an image with metadata (for example, sample type). GelBox data files integrate the raw data, supplied metadata, image adjustments, and band-level analyses in a single file to improve traceability. GelBox has a user-friendly interface and was developed using MATLAB. The software, installation instructions, and tutorials, are available at https://campbell-muscle-lab.github.io/GelBox/.

OBJECTIVE: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles.
RESULTS: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFβ1, β-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies.

The adaption of plants to stressful environments depends on long-distance responses in plant organs, which themselves are remote from sites of perception of external stimuli. Jasmonic acid (JA) and its derivatives are known to be involved in plants\' adaptation to salinity. However, to our knowledge, the transport of JAs from roots to shoots has not been studied in relation to the responses of shoots to root salt treatment. We detected a salt-induced increase in the content of JAs in the roots, xylem sap, and leaves of pea plants related to changes in transpiration. Similarities between the localization of JA and lipid transfer proteins (LTPs) around vascular tissues were detected with immunohistochemistry, while immunoblotting revealed the presence of LTPs in the xylem sap of pea plants and its increase with salinity. Furthermore, we compared the effects of exogenous MeJA and salt treatment on the accumulation of JAs in leaves and their impact on transpiration. Our results indicate that salt-induced changes in JA concentrations in roots and xylem sap are the source of accumulation of these hormones in leaves leading to associated changes in transpiration. Furthermore, they suggest the possible involvement of LTPs in the loading/unloading of JAs into/from the xylem and its xylem transport.

Literature reports suggest that the presence of proteins in pomegranate seeds is responsible for sensitization and IgE-mediated allergic reactions. The objective of this study was the analysis of a pomegranate seed extract and the isolation and characterization of proteins contained in high amounts. The extract characterization showed a protein profile with main bands at about 18 kDa and below 10 kDa upon SDS-PAGE, and molecules were recognized by specific IgEs upon immunoblotting. Then, two new 2S albumins, a monomeric and a heterodimeric one, were isolated by using classical biochemical methods. They were identified via direct protein sequencing and mass spectrometry, and their primary structure was analyzed and compared with homologous allergenic proteins via bioinformatics. In an Italian population of 703 suspected allergic patients, analyzed by using the FABER® test, the frequency of sensitization to the monomeric and heterodimeric 2S albumins was 1.7% and 0.28%, respectively. This study reports for the first time the isolation and characterization of two 2S albumins from pomegranate seeds. The clinical relevance of these molecules needs further investigation, for instance in populations having different exposures and allergy profiles.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

BACKGROUND: Anti-desmoglein (Dsg)1 is produced in pemphigus foliaceus (PF), affecting exclusively the skin. Pemphigus vulgaris (PV) shows the production of anti-Dsg3 in the mucosal form, and anti-Dsg1 and 3 in the mucocutaneous form. Anti-Dsg3 autoantibodies have been rarely reported in PF.
OBJECTIVE: To determine the factors associated with the production and pathogenicity of anti-Dsg3 in PF.
METHODS: Comparative analytical study of three patients groups: 16 PF-anti-Dsg3+, and 42 PF-anti-Dsg3(-) and 22 PV treatment-naïve cases. Serum was used in the anti-Dsg1 and 3 ELISA, and in immunoblotting (IB) with human epidermis extract. The expression of Dsg1 and 3 in paraffin sections was analyzed by immunohistochemistry (IHC). HLA-DRB1 alleles were compiled from a database.
RESULTS: In the PF-anti-Dsg3+ group: age range similar to that of the PV group (p > 0.9999); predominance of the generalized form of PF (p = 0.002); anti-Dsg3 titers lower than those of PV (p < 0.0001); IB confirmed Dsg3 identification in one (8.33%) of 12 patients; IHC showed exclusive cytoplasmic internalization of Dsg1; HLA-DRB1 alleles of susceptibility to PF, with the absence of alleles associated with PV, in the five typed patients.
CONCLUSIONS: Most of the patients in the PF-anti-Dsg3+ group were undergoing treatment.
CONCLUSIONS: The presence of anti-Dsg3 antibodies in PF was related to older age (comparable to that of PV) and the generalized form of PF. The non-pathogenicity of anti-Dsg3 antibodies in PF can be attributed to the low serum anti-Dsg3 titers, the lack of Dsg3 internalization as detected by IHC, and the absence of PV-associated HLA-DRB1 alleles.

We sought to examine how resistance exercise (RE), cycling exercise, and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation in humans. In the first study (1boutRE), younger adult men (n=8; 5±2 years of RE experience) performed a lower body RE bout with vastus lateralis (VL) biopsies obtained immediately before, 3-, and 6-hours post-exercise. In the second study (10weekRT), VL biopsies were obtained in untrained younger adults (n=36, 18 men and 18 women) before and 24 hours (24h) after their first/naïve RE bout. These participants also engaged in 10 weeks (24 sessions) of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined from a third acute cycling study (n=7) and a fourth study involving two weeks of leg immobilization (n=20, 15 men and 5 women) to determine how MyHC fragmentation was affected. In the 1boutRE study, the fragmentation of all MyHC isoforms (MyHCTotal) increased 3 hours post-RE (~ +200%, p=0.018) and returned to pre-exercise levels by 6 hours post-RE. Immunoprecipitation of MyHCTotal revealed ubiquitination levels remained unaffected at the 3- and 6-hour post-RE time points. Interestingly, a greater increase in magnitude for MyHC type IIa versus I isoform fragmentation occurred 3-hours post-RE (8.6±6.3-fold versus 2.1±0.7-fold, p=0.018). In all 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, respectively; p<0.001); however, the last RE bout response was attenuated compared to the first bout (p=0.045). The first/naïve bout response was significantly elevated in females only (p<0.001), albeit females also demonstrated a last bout attenuation response (p=0.002). Although an acute cycling bout did not alter MyHCTotal fragmentation, ~8% VL atrophy with two weeks of leg immobilization led to robust MyHCTotal fragmentation (+108%, p<0.001), and no sex-based differences were observed. In summary, RE and disuse atrophy increase MyHC protein fragmentation. A dampened response with 10 weeks of resistance training, and more refined responses in well-trained men, suggest this is an adaptive process. Given the null polyubiquitination IP findings, more research is needed to determine how MyHC fragments are processed. Moreover, further research is needed to determine how aging and disease-associated muscle atrophy affect these outcomes, and whether MyHC fragmentation is a viable surrogate for muscle protein turnover rates.