Journal: PLOS ONE

Article Title: Critical domains for NACC2-NTRK2 fusion protein activation

doi: 10.1371/journal.pone.0301730

Figure Lengend Snippet: (A) Schematic presents NACC2, NTRK2, and NACC2-NTRK2 fusion with K572R kinase dead mutation (KD). “L” refers to Leucine-rich domain, “IG1” and “IG2” refer to Immunoglobulin-like domains 1 and 2, “BTB” refers to Broad-Complex, Tramtrack and Bric a brac domain, “BEN” refers to an adapter domain found in BANP, E5R, and NACC1 proteins, “TM” refers to the Transmembrane helix, and “Kinase” refers to the tyrosine kinase domain of NTRK3. The kinase dead (KD) mutation K572R is also shown in NTRK2 and NACC2-NTRK2. Placement of domains is only approximate. (B) Representative plates of NIH3T3 cell transformation assays are shown for NTRK2, NACC2-NTRK2, and NACC2-NTRK2(KD). BCR-FGFR1 is included as positive control. In this experiment, all constructs were assayed in three independent replicates, except for NACC2-NTRK2 which was assayed six times, and BCR-FGFR1 which was assayed five times. (C) Results of NIH3T3 focus assays are presented. Each construct was assayed a minimum of three times, and the ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to BCR-FGFR1. The P value of a two-tailed paired t test comparing NACC2-NTRK2 with BCR-FGFR1 was 0.011, and is shown as * = P ≤0.05. (D) The activation of downstream signaling pathways is presented. HEK293T cell lysates expressing NTRK2, NTRK2(KD), NACC2-NTRK2, and NACC2-NTRK2(KD) were analyzed by SDS-PAGE and immunoblotted for P-NTRK (top panel), total NTRK2 (2 nd panel), P-MAPK (3 nd panel), total MAPK (4 th panel), P-PLCγ1 (5th panel, upper band), total PLCγ1(6 th panel), P-STAT3(7 th panel) and total STAT3(8 th panel). Note that in this figure, and other immunoblots of P-PLCγ1, the band of interest migrates above the 130 kD marker; the prominent band near the bottom of this blot represents antiserum cross-reactivity with the activated NTRK2 kinase domain.

Article Snippet: Genes for NACC2 (SC319661) and NTRK2 (RG221838) were purchased from Origene (Rockville, MD, USA).

Techniques: Mutagenesis, Transformation Assay, Positive Control, Construct, Two Tailed Test, Activation Assay, Protein-Protein interactions, Expressing, SDS Page, Western Blot, Marker

Journal: PLOS ONE

Article Title: Critical domains for NACC2-NTRK2 fusion protein activation

doi: 10.1371/journal.pone.0301730

Figure Lengend Snippet: (A) The sequence alignment is presented of the NACC2 BTB domain in comparison with four other BTB domains of proteins that have been structurally determined. The sequences shown include human NACC2 (UniProt Q96BF6), human NACC1 (UniProt Q96RE7), human PLZF (UniProt Q05516), human BCL6 (UniProt P41182), and human BACH1 (UniProt O148667). This alignment reveals several conserved residues shown by others to be critical for PLZF dimerization, including the D31/R45 charged pocket residues and Y86 monomer core residue [ , ]. Multi-sequence alignment was performed using Praline . (B) The structural alignment is presented showing the BTB domains of NACC2 (AlphaFold: AF-Q9DCM7-F1, blue) and PLZF (PDB: 1BUO, tan), created using Chimera. (C) A model of the NACC2 BTB domain dimer structure was created based on the PLZF dimer (PDB ID: 1BUO). The charged pocket residues D31 and R45 are indicated at the dimer interface, and the monomer core residue Y86 is also shown. (D) Lysates from HEK293T cells expressing NACC2, NACC2-NTRK2, NACC2(D31N/R45Q)-NTRK2, and NACC2(Y86A)-NTRK2 were analyzed by SDS-PAGE and immunoblotted for NACC2. Non-reducing sample buffer (left panel) or reducing sample buffer (right panel) was used for duplicate samples. Significant multimer bands of NACC2 and NACC2-NTRK2 were observed in the non-reducing conditions (left panel, lanes 2 and 3). (E) The ratio of Multimer intensity relative to Monomer intensity is shown, using intensities of three replicates quantitated by ImageJ. The P values of two-tailed paired t tests are shown: For NACC2-NTRK2 versus NACC2, P = 0.0020, indicated as ** = P ≤0.01; for NACC2-NTRK2 versus NACC2(D31N/R45Q)-NTRK2, P = 0.023, indicated as * = P ≤0.05; and for NACC2-NTRK2 versus NACC2(Y86A)-NTRK2, P = 0.0023, indicated as ** = P ≤0.01.

Article Snippet: Genes for NACC2 (SC319661) and NTRK2 (RG221838) were purchased from Origene (Rockville, MD, USA).

Techniques: Sequencing, Comparison, Residue, Expressing, SDS Page, Two Tailed Test

Journal: PLOS ONE

Article Title: Critical domains for NACC2-NTRK2 fusion protein activation

doi: 10.1371/journal.pone.0301730

Figure Lengend Snippet: (A) The interaction of NACC2 and NACC2-NTRK2 is examined. Lysates of HEK293T cells expressing NACC2-NTRK2 (designated as WT), or with the mutations D31N/R45Q or Y86A, were coexpressed with NACC2 as indicated (Lanes 4–6). Samples were prepared in E1A buffer to preserve protein-protein interactions. The 3 rd panel presents the results of immunoprecipitation using antibodies against NTRK2, to recover the NACC2-NTRK fusion proteins, followed by immunoblotting to detect NACC2 present in the immune complexes. Lane 4 clearly shows binding of NACC2 to NACC2-NTRK2, which is reduced in the presence of the mutations D31N/R45Q (lane 5), or Y86A (lane 6). Control immunoblots are shown for NTRK2 (top panel) and NACC2 (middle panel). (B) The ratio of the intensity of NACC2 relative to the NACC2-NTRK2 fusion with which it was co-immunoprecipitated is shown, using intensities of multiple replicates quantitated by ImageJ. The P values of two-tailed paired t tests are shown: For NACC2 bound to NACC2-NTRK2 versus NACC2(D31N/R45Q)-NTRK2, P = 0.034, indicated as * = P ≤0.05; for NACC2 bound to NACC2-NTRK2 versus NACC2(Y86A)-NTRK2, P = 0.003, indicated as ** = P ≤0.01. (C) HEK293T cells expressing NACC2-NTRK2, NACC2-NTRK2(KD), NACC2(D31N/R45Q)-NTRK2, NACC2(Y86A)-NTRK2 or NACC2(ΔBTB)-NTRK2 were examined for the activation of downstream signaling pathways. Cell lysates were analyzed by SDS-PAGE and immunoblotted for P-NTRK (top panel), total NTRK2 (2 nd panel), P-MAPK (3 nd panel), total MAPK (4 th panel), P-PLCγ1 (5th panel, upper band), total PLCγ1 (6 th panel), P-STAT3(7 th panel) and total STAT3(8 th panel). (D) The immunoblots presented in (C), together with additional independent replicates, were quantitated by ImageJ and used to calculate the changes in P-NTRK2, P-MAPK, P-PLCγ1, and P-STAT3, after normalization of each sample in comparison to total NTRK2, MAPK, PLCγ1, and STAT3. The P values of two-tailed paired t tests are shown for wild-type NACC2-NTRK2 versus each of the mutants KD, D31N/R45Q, Y86A, and ΔBTB. Statistical significance is indicated as follows: ns = not significant; * = P ≤0.05; ** = P ≤0.01; *** = P ≤0.001; **** = P ≤0.0001. Four independent replicates were used to calculate the changes in P-NTRK2 relative to NTRK2, and three independent replicates were used for changes in P-MAPK, P-PLCγ1, and P-STAT3. (E) The BTB domain mutants of NACC2-NTRK2 were examined for NIH3T3 cell transformation activity. In this experiment, all constructs were assayed in three independent replicates, except for NACC2-NTRK2 which was assayed six times. The ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to NACC2-NTRK2, which was set to 100%.

Article Snippet: Genes for NACC2 (SC319661) and NTRK2 (RG221838) were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Protein-Protein interactions, Immunoprecipitation, Western Blot, Binding Assay, Control, Two Tailed Test, Activation Assay, SDS Page, Comparison, Transformation Assay, Activity Assay, Construct

Journal: PLOS ONE

Article Title: Critical domains for NACC2-NTRK2 fusion protein activation

doi: 10.1371/journal.pone.0301730

Figure Lengend Snippet: (A) NACC2-NTRK2 is shown schematically with the BTB domain from residues 20–120, a partial BEN domain at residues 351–418, and the disorder region (DR) in between. Also shown in comparison is the deletion of residues 120–418 of NACC2, which retains only the N-terminal BTB domain, designated BTB-NTRK2. (B) Downstream activation by BTB-NTRK2. HEK293T cells expressing NACC2-NTRK2 or BTB-NTRK2 were examined for activation of downstream signaling pathways, as described previously for Figs and . (C) The immunoblots presented in (B), together with additional independent replicates, were quantitated by ImageJ and used to calculate the changes in P-NTRK2, P-MAPK, P-PLCγ1, and P-STAT3, after normalization of each sample in comparison to total NTRK2, MAPK, PLCγ1, and STAT3. The P values of two-tailed paired t tests are shown for wild-type NACC2-NTRK2 versus BTB-NTRK2. Statistical significance is indicated as follows: ns = not significant; * = P ≤0.05; ** = P ≤0.01. Three independent replicates were used for each condition, except for P-STAT3 for which five independent replicates were available. (D) Lysates of HEK293T cells expressing NACC2-NTRK2 or BTB-NTRK2 were examined for homo-multimerization. Samples were loaded using non-reducing sample buffer (left panel), or reducing sample buffer containing β-mercaptoethanol (right panel), and then analyzed by SDS-PAGE and immunoblotted for NTRK2. (E) Hetero-multimerization assay of NACC2-NTRK2 and BTB-NTRK2. Fusion clones were cotransfected with NACC2 (lanes 4 and 5) and examined for their ability to bind to NACC2 in an immune complex prepared using antibodies against NTRK2. The 3 rd panel shows clearly the binding of NACC2 to NACC2-NTRK2 (lane 4), indicated by a red arrowhead. NACC2 also clearly binds to BTB-NTRK2 (lane 5), again indicated by a red arrowhead. Control lysate blots are shown for NTRK2 (1 st panel) and NACC2 (2 nd panel). Control IP immunoblot is shown in 4 th panel for NTRK2. (F) The BTB-NTRK2 fusion was compared with NACC2-NTRK2 in NIH3T3 cells transformation assays. In this experiment, NACC2-NTRK2 was assayed in six independent replicates, and BTB-NTRK2 was assayed three times. The ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to NACC2-NTRK2, which was set to 100%. The P value of a two-tailed paired t test comparing NACC2-NTRK2 with BTB-NTRK2 was 0.00091, and is shown as *** = P ≤0.001.

Article Snippet: Genes for NACC2 (SC319661) and NTRK2 (RG221838) were purchased from Origene (Rockville, MD, USA).

Techniques: Comparison, Activation Assay, Expressing, Protein-Protein interactions, Western Blot, Two Tailed Test, SDS Page, Clone Assay, Binding Assay, Control, Transformation Assay

Journal: PLOS ONE

Article Title: Critical domains for NACC2-NTRK2 fusion protein activation

doi: 10.1371/journal.pone.0301730

Figure Lengend Snippet: (A) NACC2-NTRK2 examined thus far in this work is shown as NACC2-NTRK2(ex4:ex13), in comparison with two derivatives: NACC2-NTRK2-ΔTM, deleting just the transmembrane helix, and NACC2-NTRK2(ex4:ex15) which deletes NTRK2 exons 13 and 14. (B) HEK293T cells expressing NACC2-NTRK2, NACC2-NTRK2-ΔTM and NACC2-NTRK2(ex4:ex15) were examined for activation of downstream signaling pathways, as described previously for Figs and . (C) The immunoblots presented in (B), together with additional independent replicates, were quantitated by ImageJ and used to calculate the changes in P-NTRK2, P-MAPK, P-PLCγ1, and P-STAT3, after normalization of each sample in comparison to total NTRK2, MAPK, PLCγ1, and STAT3. The P values of two-tailed paired t tests are shown for wild-type NACC2-NTRK2 versus each of the mutants KD and ΔTM. Statistical significance is indicated as follows: ns = not significant; * = P ≤0.05; ** = P ≤0.01; *** = P ≤0.001; **** = P ≤0.0001. Three independent replicates were used for each condition. (D) The biological activity of NACC2-NTRK2(ex4:ex13) was compared to NACC2-NTRK2-ΔTM using NIH3T3 cell transformation assays. In this experiment, both NACC2-NTRK2(ex4:ex13) and NACC2-NTRK2-ΔTM were assayed in six independent replicates. The ratio of foci/G418-resistant colonies was calculated as a percentage of transformation +/- SEM relative to NACC2-NTRK2(ex4:ex13), which was set to 100%. The P value of a two-tailed paired t test comparing NACC2-NTRK2(ex4:ex13) with NACC2-NTRK2-ΔTM was 0.0096, and is shown as ** = P ≤0.01. (E) Lysates prepared from cells expressing either NACC2-NTRK2 (left lanes) or NACC2-NTRK2-ΔTM (right lanes) and treated with cycloheximide for the indicated time points were examined by SDS-PAGE and immunoblotted for NTRK2 The left lanes show decreasing amounts of NACC2-NTRK2 in the presence of cycloheximide, while the right lanes reveal little or no change in NACC2-NTRK2-ΔTM under identical conditions. (F) The half-life of NACC2-NTRK2(ex4:13) was determined to be approximately 18 hours, that of NACC2-NTRK2(ex4:15) approximately 42 hours, while the half-life of NACC2-NTRK2-ΔTM could not be determined due to its stability over the time course of this experiment. Quantitation was accomplished using ImageJ with a minimum of three replicates for each sample, and half-lives were determined by least squares analysis.

Article Snippet: Genes for NACC2 (SC319661) and NTRK2 (RG221838) were purchased from Origene (Rockville, MD, USA).

Techniques: Comparison, Expressing, Activation Assay, Protein-Protein interactions, Western Blot, Two Tailed Test, Activity Assay, Transformation Assay, SDS Page, Quantitation Assay

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