Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet:

Article Snippet: dCas9-fusion + sgRNA library expression vector (see Supplementary File 2) , , Addgene #171798.

Techniques: Virus, Synthesized, Recombinant, Plasmid Preparation, Expressing, Software, Microarray

Journal: The Plant Journal

Article Title: Catch & Release—rapid cost‐effective protein purification from plants using a DIY GFP ‐Trap‐protease approach

doi: 10.1111/tpj.70544

Figure Lengend Snippet: Functional characterization of KEA1 constructs using Catch & Release vectors. (A) Photographs and PAM images ( F v / F m ) comparing WT, kea1‐1kea2‐1 , and complemented kea1‐1kea2‐1 lines expressing FAST‐Green (FG)‐KEA1‐TEV‐mVenus, FAST‐Red (FR)‐KEA1‐TEV‐mVenus, FAST‐Red‐KEA1‐3C‐mVenus, and FAST‐Red‐KEA1‐mCherry‐TEV‐mVenus using a UBQ10 promoter. Scale bar = 1 cm. (B) F v / F m in WT, kea1‐1kea2‐1 , and complemented plants expressing FAST‐Green (FG)‐KEA1‐TEV‐mVenus, FAST‐Red‐KEA1‐TEV‐mVenus, FAST‐Red‐KEA1‐3C‐mVenus, and FAST‐Red‐KEA1‐mCherry‐TEV‐mVenus. Data are presented as mean ± SEM ( n = 14). Statistical significance was determined via one‐way anova , with different letters indicating significantly different groups. (C) Confocal laser micrographs of Arabidopsis protoplasts expressing KEA1‐TEV‐mVenus, KEA1‐3C‐mVenus, and KEA1‐mCherry‐TEV‐mVenus. Fluorescence images show KEA1‐mVenus (green, left), KEA1‐mCherry (cyan, middle left), chlorophyll autofluorescence (red, middle right), and a merged view (right). Scale bar = 10 μm. (D) Immunoblot analysis of protease site accessibility in whole‐leaf extracts from WT and complemented kea1kea2 plants expressing KEA1‐3C‐mVenus, KEA1‐TEV‐mVenus, KEA1‐TEV‐mCherry, and KEA1‐mCherry‐TEV‐mVenus. The same membrane was stained with Coomassie brilliant blue (c.b.b.) and rubisco large subunit (rbcL) was used as a loading control. The exact same protein extracts were used for pre‐ and post‐protease treatment samples. (E) Immunoblot analysis of protease site accessibility in Nicotiana benthamiana leaves infiltrated with KEA1‐TEV‐Strep, KEA1‐TEV‐Flag, KEA1‐TEV‐MYC, and KEA1‐TEV‐HA. Un‐infiltrated leaves served as WT controls. The same membrane was stained with Coomassie brilliant blue (c.b.b.) and rbcL was used as a loading control. The exact same proteins extracts were used for pre‐ and post‐protease treatment samples.

Article Snippet: TEV protease , Addgene ID: 171782.

Techniques: Functional Assay, Construct, Expressing, Fluorescence, Western Blot, Membrane, Staining, Control

Journal: The Plant Journal

Article Title: Catch & Release—rapid cost‐effective protein purification from plants using a DIY GFP ‐Trap‐protease approach

doi: 10.1111/tpj.70544

Figure Lengend Snippet: Isolation and structural features of KEA1 from Arabidopsis thaliana . (A) Schematic overview of the protein purification workflow using the homemade GFP‐Trap and proteases. Arabidopsis thaliana plants were first ground to powder in liquid nitrogen and solubilized for 30 min. The solubilized protein mixture was then incubated with homemade GFP‐Trap for 1–4 h at 4°C, followed by multiple wash steps. The protein was released by on‐resin protease digestion, resulting in an elution containing both the non‐tagged target protein and the protease. For information on volumes please refer to the section. (B) SDS‐PAGE analysis verifying successful KEA1 purification from stable A. thaliana lines, using the described workflow. Lanes represent: Total protein lysate (Input, Inp.), unbound fraction after incubation with GFP‐Trap (Flow‐Through, FT), affinity‐bound protein before protease cleavage (b. TEV), protein eluted after TEV cleavage (Elution, Elu.), and the GFP‐Trap resin after elution (beads). C.b.b. indicates that the gel was stained with Coomassie brilliant blue. (C) Schematic representation of the plastid inner envelope potassium cation efflux antiporter KEA1. The N‐terminal region is predicted to contain 2–3 coiled‐coil domains (depicted as helical structures), while the C‐terminal domain, facing the stroma, is fluorescently tagged and includes the KTN domain.

Article Snippet: TEV protease , Addgene ID: 171782.

Techniques: Isolation, Protein Purification, Incubation, SDS Page, Purification, Staining

Journal: The Plant Journal

Article Title: Catch & Release—rapid cost‐effective protein purification from plants using a DIY GFP ‐Trap‐protease approach

doi: 10.1111/tpj.70544

Figure Lengend Snippet: Double‐fluorescent tag purification and characterization of PGDH3‐mCherry. (A) Schematic representation of the ‘Catch & Release’ purification workflow using the double‐fluorescent tag. Transiently protein expressing Nicotiana benthamiana leaves were ground, solubilized, and incubated with GFP‐Trap resin to capture the fusion protein, followed by a series of wash steps. On‐resin digestion with a His‐tagged protease then releases the mCherry‐tagged target protein from the beads. The eluate, containing both the target protein and the protease, is subjected to immobilized metal affinity chromatography (IMAC). This step removes the His‐tagged protease, resulting in a final sample of pure, protease‐free mCherry‐tagged target protein. For information on volumes please refer to the section. (B) Time‐resolved elution of PGDH3‐mCherry‐TEV‐mVenus. The left image depicts the fluorescence of PGDH3 bound to GFP resin before protease digestion. After enzymatic cleavage, eluted protein (free of resin) was collected at different time points and imaged under a fluorescence stereoscope. PGDH3‐mCherry‐TEV‐mVenus was transiently expressed utilizing the Catch & Release vectors including the UBQ10 promoter. (C) SDS‐PAGE analysis of elution fractions from (B), stained with Coomassie brilliant blue (c.b.b.). TEV protease served as a loading control. (D) SDS‐PAGE analysis stained with Coomassie brilliant blue (c.b.b.) illustrating PGDH3‐mCherry purification, including removal of His‐tagged TEV protease using Ni‐NTA affinity chromatography (IMAC). Lanes represent: Total protein lysate (Input, Inp.); unbound fraction after GFP‐Trap incubation (Flow‐Through, FT); protein eluted from GFP‐Trap after TEV cleavage (Elution Trap); final purified protein after IMAC step (Elution IMAC); GFP‐Trap resin post‐elution (beads Trap); IMAC resin post‐elution (beads IMAC). (E) Mass distribution histogram of purified PGDH3‐mCherry obtained using the isolation protocol. The histogram represents mean trajectory contrasts detected in a dynamic mass photometry analysis ( n = 1 movie, 1 min), including trajectories of at least 151 ms in length ( n = 2847 trajectories). Percentages correspond to the fraction of counts for each peak. Predicted 3D structures of both isoforms were generated using AlphaFold 3.0 (Abramson et al., ). (F) Michaelis‐Menten kinetics of purified PGDH3‐mCherry, assessing phosphoglycerate dehydrogenase function. Data are presented as mean ± SEM ( n = 3).

Article Snippet: TEV protease , Addgene ID: 171782.

Techniques: Purification, Expressing, Incubation, Affinity Chromatography, Fluorescence, SDS Page, Staining, Control, Isolation, Generated

Journal: Frontiers in Medicine

Article Title: Hyperbaric oxygen therapy and N-acetylcysteine: a redox-dependent interaction

doi: 10.3389/fmed.2026.1829074

Figure Lengend Snippet: Context-dependent effects of combined N-acetylcysteine (NAC) and hyperbaric oxygen therapy (HBOT). The effects of NAC in combination with HBOT depend on baseline redox status, dose, and timing of administration. When given prior to HBOT, NAC may suppress the initial ROS burst required for adaptive signaling, thereby attenuating HBOT efficacy. In contrast, under conditions of elevated oxidative stress or when administered after injury, NAC can reduce excessive ROS, while preserving HBOT-induced signaling, resulting in adaptive and protective responses.

Article Snippet: Cermik et al. ( ) , Acetaminophen-induced nephrotoxicity in Sprague - Dawley rats *(NAC, NAC + HBOT groups) , NAC, 100 mg/kg i.p. once daily and HBOT exposure at 2.8 ATA, 90 min, 2 sessions per day, for the consecutive 5 days. NAC and HBOT were administered 24 h after injury induction. NAC was administered during the same experimental period as HBOT. , NAC treatment significantly attenuated biochemical markers of renal injury and reduced inflammatory mediators. Importantly, the combined NAC and HBOT produced more pronounced protective effects than NAC alone, resulting in the lowest creatinine, urea, cytokine, and neopterin levels, as well as improved renal histology..

Techniques: Preserving