Journal: iScience

Article Title: β-catenin links cell seeding density to global gene expression during mouse embryonic stem cell differentiation

doi: 10.1016/j.isci.2021.103541

Figure Lengend Snippet:

Article Snippet: J1 Mouse Embryonic Stem Cells , ATCC , ATCC SCRC-1010.

Techniques: Virus, Recombinant, Transfection, Staining, Protease Inhibitor, SYBR Green Assay, Western Blot, cDNA Synthesis, Cloning, shRNA, Control, Plasmid Preparation, Software, Microscopy

Journal: Frontiers in Cellular Neuroscience

Article Title: Adipose-derived stem cell-conditioned medium mitigates ischemia-induced neuronal injury via the JAK1/STAT3 signaling pathway

doi: 10.3389/fncel.2026.1744887

Figure Lengend Snippet: ADSC-CM promotes neurite outgrowth in OGD-injured neurons through the JAK1-STAT3 signaling pathway. (A) On day 5 of cultivation, neuronal neurites extended and formed an extensive neural network. Immunofluorescence staining showed that the neurons were positive for the neuronal-specific marker Tuj1. After OGD injury, neuronal neurites were damaged, partially interrupted, and disappeared. (B) Western blot bands of JAK1, pJAK1, STAT3, pSTAT3, and GAPDH for each group. (C) Comparison of the relative ratio of pJAK1 to JAK1 across experimental groups ( n = 4). (D) Comparison of the relative ratio of pSTAT3 to STAT3 across experimental groups ( n = 4). (E) Immunofluorescence images of neurons from each group, showing the effects of ADSC-CM and GLPG0634 on neurite outgrowth. Neurons were stained with the Tuj1 antibody (red) to label the neuronal cytoskeleton, and nuclei were counterstained with DAPI (blue). (F) Diagram illustrating how to measure the length of the longest neurite and the number of primary neurites in neurons. The green line shows the trajectory of the longest neurite, and white arrows point to primary neurites. (G,H) Comparison of the length of the longest neurite and the number of primary neurites in neurons across experimental groups ( n = 4). Data are expressed as means ± SEM. The difference between the groups was assessed using a one-way ANOVA followed by Bonferroni post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 100 μm (A) , 20 μm (E,F) .

Article Snippet: After overnight incubation with rabbit anti-Tuj1 antibody (1:200, BM3881, Boster, Wuhan, China) at 4 °C, cells were washed and then incubated with Cy3-conjugated goat anti-rabbit IgG (1:400, AS007, ABclonal) for 1 h at room temperature in the dark.

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Comparison

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Chemical structure of standard Cbls with their corresponding β-axial groups shown. b , Co-crystal structure of the env8 -OHCbl complex (PDB 4FRG 19 ). Global RNA architecture (left) is displayed as cartoon, nucleotides critical for recognition of the variable β-axial group are shown in cyan (A68), green (A20), and yellow (G19), and OHCbl is represented by magenta van der Waals spheres. The ligand binding pocket (right) consists of a π-stacking network between nucleotides G19-A20 and A20-A68 that is unimpeded by the small (-OH) β-axial group. c , Simplified chemical structures of previously characterized 20 β-axial-modified Cbls 4 - 7 tested against env8 . d , Reduction-free synthetic route 25 , 26 to make Cbl derivatives from CNCbl using a variable alkyne. e , Overview of our expanded β-axial-modified Cbl library.

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Ligand Binding Assay, Modification

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Binding curve of the fluorescence induction of CNCbl-5×PEG-ATTO590 binding to env8 . b , Representative displacement binding curve for MeCbl and Cbl 4 . Binding curves are shown as mean and s.e.m. from independent experiments ( n = 3). K D values are reported as mean and s.d. from independent experiments ( n = 3). c , Log-transformed K D values for 1 - 44 shown as mean and s.d. from independent experiments ( n = 4 except for data from 1 , 2 , 4 - 7 , 10 , 14 , 19 , 41 , and 42 where n = 3 and data from 21 , 36 , and 37 where n = 5.). d , Locations of the training and test set from the Q 2 -focused modeling in two-dimensional chemical space constructed from PC1 and PC2 of the whole data set. e , Measured ln [K D ] values plotted with the value predicted by the Q 2 -focused model. f , Overview of our lead compound screen from a 513-compound alkyne library using our binding-based models. g , Simplified chemical structures of representative derivatives that our binding-based models predict to be tight ( 45 - 48 ), moderate ( 49 and 50 ), and weak ( 51 - 53 ) binders. h , Measured ln [K D ] values plotted with the value predicted by our binding-based models. The experimental data are represented as mean and s.d. from independent experiments ( n = 3 except for data from 45 , 48 , and 53 where n = 4) and the predicted data are shown as the mean and s.d. from independent predictions ( n = 3) from our three models (baseline, R 2 -focused, and Q 2 -focused).

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: RNA Binding Assay, Binding Assay, Fluorescence, Transformation Assay, Construct

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Box-and-whisker plot of the OD 600 -normalized relative fluorescence units (RFU) of E. coli cells with and without reporter plasmid ( env8 -GFPuv) and ligand (MeCbl and Cbl 4 ) added. Data are shown from independent experiments ( n = 4). b , Fold repression (defined as the ratio of “(−)-Cbl RFU” and “(+)-Cbl RFU” values) 33 for our env8 reporter system in the presence of Cbl 1 - 44 shown as mean and s.d. from biological replicates ( n = 4 except for data from 6 where n = 3). c , Plot comparing the log-transformed fold repression and K D data. d , Locations of the training and test set from the Q 2 -focused modeling in two-dimensional chemical space constructed from PC1 and PC2 of the whole data set. e , Measured fold repression values plotted with the value predicted by the Q 2 -focused model. f , Overview of our lead compound screen from a 513-compound alkyne library using our function-based models. g , Simplified chemical structures of representative derivatives that our function-based models predict to be strong ( 54 - 57 ), moderate ( 58 and 59 ), and weak ( 60 - 62 ) repressors. h , Measured fold repression values plotted with the value predicted by our function-based models. The experimental data are represented as mean and s.d. from biological replicates ( n = 4 except for data from 58 , 59 , 61 , and 62 where n = 3) and the predicted data are shown as the mean and s.d. from independent predictions ( n = 3) from our three models (i.e., baseline, R 2 -focused, and Q 2 -focused).

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Activity Assay, Whisker Assay, Fluorescence, Plasmid Preparation, Transformation Assay, Construct

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Representative MD starting states of the env8 -CNCbl and env8 -Cbl 4 complexes from one (of three) independent 1 μs trajectory. The A20(C6)-G19(C6) and A20(C6)-A68(N7) distances are shown as double arrows. In all structural representations, the binding pocket nucleotides G19 (yellow), A20 (green), and A68 (cyan) are numbered in reference to full-length env8 and colored, the β-axial group is shown in magenta, and dashed lines represent proposed hydrogen bonds. b , The average A20(C6)-G19(C6) (left) and A20(C6)-A68(N7) (right) distances measured over the course of three independent MD trajectories for both env8 -CNCbl and env8 -Cbl 4 complexes. c , Co-crystal structure of env2 -CNCbl showing only the binding pocket nucleotides and the β-axial group. All mesh representations correspond to a simulated annealing 2F o -F c map where A20 and the ligand were omitted from the model and are shown at 1 σ contour. d , Same as in c but for env2 -Cbl 4 . e , Same as in c but for env2 -Cbl 32 (left) and showing a van der Waal sphere representation of the binding pocket (right). f , Same as in e but for env2 -Cbl 29 . g , Same as in c but for env2 -Cbl 42 . h , Schematic representation of design hypotheses that emerge from our 11 RNA-ligand co-crystal structures.

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Binding Assay

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Modified click reaction 25 to make Cbl derivatives from 58 using a variable azide. b , Simplified chemical structures of Cbls 63 - 65 that were made to systematically test our design hypotheses. c , Fluorescence displacement binding curve for CNCbl and 63 - 65 shown as mean and s.e.m. from independent experiments ( n = 4 except for data from CNCbl where n = 3). K D values are reported as mean and s.d. from independent experiments ( n = 4 except for data from CNCbl where n = 3). d , To verify the near-picomolar binding of 63 and 64 , we used ITC with the env8 aptamer domain. Given that this RNA shows weaker ligand binding than the full-length env8 , 19 it is in the perfect regime for ITC. Representative ITC binding isotherm for the env8 aptamer domain with CNCbl, 63 , and 64 . e , Co-crystal structure of env2 -Cbl 63 showing only the binding pocket nucleotides and the β-axial group. Proposed hydrogen bonds, which are weakly supported by the electron density, are shown as double arrows. All mesh representations correspond to a simulated annealing 2F o -F c map where A20 and the ligand were omitted from the model and are shown at 1 σ contour. f , Same as in e but for env2 -Cbl 64 . g , Fold repression for our reporter system in the presence of CNCbl and 63 - 65 and for cells with CNCbl and excess amounts of 63 - 65 added. Mean and s.d. from biological replicates ( n = 4) are shown. For comparison, the mean fold repression of 10 nM CNCbl is shown as a dashed line its s.d. is represented as a shaded box. h , Simplified chemical structure of the titratable pyridine Cbl 66 . i , Representative ITC binding isotherms for env8 aptamer domain with CNCbl, 29 , and 66 at pH 8 and pH 5. For all ITC data, K D values are reported as mean and s.d. from independent experiments ( n = 3).

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Modification, Fluorescence, Binding Assay, Ligand Binding Assay, Comparison

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Chemical structure of the eight hits that emerged from our structure-informed docking, with their biphenyl-like scaffold highlighted in gray. b , TO-based displacement assay to identify env8 binding compounds that induce fluorescence attenuation. This was used as an orthogonal binding experiment because we observed ligand-induced fluorescence induction of CNCbl-5×PEG-ATTO590. c , MST trace of fluorescently labeled env8 alone and in the presence of CNCbl, 68 , and 71 . All ligands induced an observable change to the MST trace of env8 suggestive of binding. d , TO-displacement binding curve for CNCbl, 68 , and 71 shown as mean and s.d. from independent experiments ( n = 3). K D values are reported as mean and s.d. from independent experiments ( n = 3). e , Native polyacrylamide gel showing a gradual shift from a slower migrating, extended conformation to a faster migrating, kissing-loop conformation of env8 upon titration of CNCbl (0.01–10 μM). Data are shown from an individual experiment. f , Same as in e but after addition of CNCbl, 68 , and/or 71 to demonstrate competitive binding of 68 and 71 . Similar results were obtained from repeated experiments ( n = 8 for CNCbl, n = 3 for 68, and n = 2 for 71). g , MST trace from a competition experiment where env8 -CNCbl was monitored with and without the addition of excess amounts of 68 or 71 . Both ligands induced an observable change to the MST trace of env8 -CNCbl suggestive of competitive binding. All MST data are shown as representative traces from multiple independent experiments ( n = 4). h , Top-ranked docking poses of 68 and 71 to env2 in the A20-out and A20-in state.

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Binding Assay, Fluorescence, Labeling, Titration

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Schematic representation of the secondary structures of full-length env8 , env8 aptamer domain, and the env8 and env2 RNA crystallography constructs. The binding pocket nucleotides G19 (yellow), A20 (green), and A68 (cyan) are colored and the sites of variation between the env8 and env2 RNA sequences are indicated in magenta. Other changes to the env8 wild-type RNA sequence are the insertion of a gGAAAc tetraloop at L5, the variable lengths of helices P1 and P5 (either four or six base pairs, bp), and a 3′-end adenosine, which were all incorporated to aid crystallization. b , Binding curve of the fluorescence induction of CNCbl-5×PEG-ATTO590 binding to env8 aptamer domain and env2 crystal construct shown as mean and s.e.m. from independent experiments ( n = 3). The K D values are reported as mean and s.d. from independent experiments ( n = 3). c , Fold repression values for our env8 and env2 reporter systems in the presence of 10 μM CNCbl, reported as mean and s.d. from biological replicates ( n = 4). d , Co-crystal structure of env2 -CNCbl aligned to the env8 -OHCbl complex (PDB 4FRG 19 ). Global RNA architecture (left) is displayed as cartoon, with sites of variation between the env8 and env2 shown in magenta. The ligand binding pocket (right) is shown in sticks and shows very strong agreement between the two RNA-ligand complexes (RMSD = 0.45 Å). Given that env2 shows near-identical ligand binding, regulatory activity, and local and global RNA architecture, all structural information from env2 can be translated to env8 .

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Biomarker Discovery, Construct, Binding Assay, Sequencing, Crystallization Assay, Fluorescence, Ligand Binding Assay, Activity Assay

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Published DNAJB2 mutations

Article Snippet: Blots were stained with the following primary antibodies: calnexin Rb mAb C5C9 (Cell Signaling Technology, 2679, RRID:AB_2228381); DNAJB2 Rb pAb (Proteintech, 10 838-1-AP, RRID:AB_2277491); DNAJB6 Rb mAb [EPR17122] (Abcam, ab198995, RRID:AB_2924896); histone 3 Rb pAb (Abcam, ab1791, RRID:AB_302613); tubulin Rt mAb YL1/2 (Abcam, ab6160, RRID:AB_305328); V5 Ms mAb (Thermo Fisher, R960-25, RRID:AB_2556564); V5 Rb mAb D3H8Q (Cell Signaling Technology, 13202, RRID:AB_2687461).

Techniques:

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Patients and clinical findings. ( A ) Pedigree of our family. The DNAJB2 genotype (+, wild-type; ext, c.832 T > G p. * 278Glyext * 83) is shown for the family members available for genetic analysis. ( B ) Muscle MRIs of the proband (left) show diffuse neurogenic degenerative change in the soleus and gastrocnemius lateralis muscles of the lower legs but also myopathic-dystrophic fatty replacement focal changes in anterior parts of gluteus minimus and left soleus (arrows). Degenerative changes in the younger brother (right) are similar but milder with more neurogenic but also spots of myopathic replacement in the left adductor magnus (arrow) and the outer part of both peroneus longus muscles. ( C ) Gastrocnemius muscle biopsy of the proband (1 and 2 haematoxylin/eosin, 3 NADH and 4 ATPase pH 4.6) showing classical neurogenic changes such as fibre type grouping and groups of atrophic fibres together with clear myopathic changes such as rimmed vacuoles (arrowhead in 1), fibre splitting (arrowhead in 2) and heavily increased number of internalized myonuclei. Furthermore, in NADH staining , some small dark angulated fibres (black arrowhead in 3) and few moth-eaten fibres (white arrowhead in 3) are found. Scale bars 25 μm for 1 and 2, 100 μm for 3 and 4.

Article Snippet: Blots were stained with the following primary antibodies: calnexin Rb mAb C5C9 (Cell Signaling Technology, 2679, RRID:AB_2228381); DNAJB2 Rb pAb (Proteintech, 10 838-1-AP, RRID:AB_2277491); DNAJB6 Rb mAb [EPR17122] (Abcam, ab198995, RRID:AB_2924896); histone 3 Rb pAb (Abcam, ab1791, RRID:AB_302613); tubulin Rt mAb YL1/2 (Abcam, ab6160, RRID:AB_305328); V5 Ms mAb (Thermo Fisher, R960-25, RRID:AB_2556564); V5 Rb mAb D3H8Q (Cell Signaling Technology, 13202, RRID:AB_2687461).

Techniques: Muscles, Staining

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: The DNAJB2 mutation. ( A ) The T > G change identified in the proband affects the termination codon of the DNAJB2 transcript variant 1 (NM_001039550.2) and is predicted to cause a C-terminal extension of the DNAJB2a protein isoform (NP_001034639.1:p. * 278Glyext * 83). In variant 2 (NM_006736.6) encoding the DNAJB2b isoform, the altered nucleotide lies within the 3′ UTR and does not affect the protein product. In the diagram of the DNAJB2 transcript, the non-coding regions are shown in grey, the normal coding regions in black and the extended open reading frame caused by the mutation in magenta. Both the DNAJB2a (top) and DNAJB2b (bottom) proteins contain an N-terminal J domain (JD; orange) followed by a glycine/phenylalanine-rich region (G/F; blue), and a C-terminal domain (CTD; yellow) containing a serine-rich region (SR) and two ubiquitin-interacting motifs (UIMs). The two isoforms differ in their C-terminal parts (green), and DNAJB2b has a C-terminal geranylgeranyl moiety (GG) anchoring it to the endoplasmic reticulum. The extended DNAJB2a protein (p. * 278Glyext * 83) produced from the mutant allele has a C-terminal extension of 83 amino acids (magenta; sequence shown in the box). ( B ) RNA sequencing (RNAseq) coverage graphs covering the last exon(s) of the DNAJB2 transcripts. RNAseq of the proband (P) muscle sample showed equal expression of the wild-type and mutant alleles (arrow) and did not indicate splicing changes or altered isoform ratio compared with other samples run in the same batch (C1–5).

Article Snippet: Blots were stained with the following primary antibodies: calnexin Rb mAb C5C9 (Cell Signaling Technology, 2679, RRID:AB_2228381); DNAJB2 Rb pAb (Proteintech, 10 838-1-AP, RRID:AB_2277491); DNAJB6 Rb mAb [EPR17122] (Abcam, ab198995, RRID:AB_2924896); histone 3 Rb pAb (Abcam, ab1791, RRID:AB_302613); tubulin Rt mAb YL1/2 (Abcam, ab6160, RRID:AB_305328); V5 Ms mAb (Thermo Fisher, R960-25, RRID:AB_2556564); V5 Rb mAb D3H8Q (Cell Signaling Technology, 13202, RRID:AB_2687461).

Techniques: Mutagenesis, Variant Assay, Ubiquitin Proteomics, Produced, Sequencing, RNA Sequencing, Expressing

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Western blotting of patient biopsy. ( A ) Western blotting of a muscle biopsy from the proband (P) revealed a reduced amount of both DNAJB2a and DNAJB2b proteins compared with pooled control (C), and no detectable mutant protein. P1 and P2 are independently prepared samples from the same biopsy. Post-blotting Coomassie staining of the myosin heavy chain (MYHC CBB) is shown as loading control. ( B ) Control (C1, C2) and proband (P) muscle biopsies were fractionated with the ProteoExtract Subcellular Proteome Extraction Kit to cytosolic (F1), membrane/organelle (F2), nuclear (F3) and cytoskeletal/insoluble (F4) fractions and analysed by western blotting. Total protein, tubulin, calnexin and histone 3 are shown as loading and fractionation controls. ( C ) The levels of DNAJB2 relative to total protein were quantified from the F1 and F2 fractions in (B) and represented normalized to the mean of control samples. Both DNAJB2 isoforms showed a ~50% reduction in the biopsy of the proband.

Article Snippet: Blots were stained with the following primary antibodies: calnexin Rb mAb C5C9 (Cell Signaling Technology, 2679, RRID:AB_2228381); DNAJB2 Rb pAb (Proteintech, 10 838-1-AP, RRID:AB_2277491); DNAJB6 Rb mAb [EPR17122] (Abcam, ab198995, RRID:AB_2924896); histone 3 Rb pAb (Abcam, ab1791, RRID:AB_302613); tubulin Rt mAb YL1/2 (Abcam, ab6160, RRID:AB_305328); V5 Ms mAb (Thermo Fisher, R960-25, RRID:AB_2556564); V5 Rb mAb D3H8Q (Cell Signaling Technology, 13202, RRID:AB_2687461).

Techniques: Western Blot, Control, Mutagenesis, Staining, Extraction, Membrane, Fractionation

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Membrane localization of mutant DNAJB2. ( A ) In silico prediction. The amino acid sequence of DNAJB2a p. * 278Glyext * 83 was analysed with transmembrane helix prediction algorithms. The graph shows the scores from TMHMM (orange trace) and TMPRED (blue solid trace, in–out orientation, dashed trace, out–in) for the 83 amino acid extension. ( B ) Stably transfected C2C12 myotubes induced to express wild-type or p. * 278Glyext * 83 (ext) DNAJB2a, and non-induced control cells, were fractionated with the ProteoExtract Subcellular Proteome Extraction Kit to cytosolic (F1), membrane/organelle (F2), nuclear (F3) and cytoskeletal/insoluble (F4) fractions. Endogenous and overexpressed DNAJB2a were predominantly cytosolic, whereas p. * 278Glyext * 83 was enriched in the membrane fraction similarly to endogenous DNAJB2b. ( C ) T-REx 293 cells were transfected with a combination of wild-type DNAJB2a and DNAJB2b (a + b wt) or DNAJB2a p. * 278Glyext * 83 (ext) and fractionated. Wild-type DNAJB2a was mostly found in the cytosolic (CYT) fraction, whereas DNAJB2b and p. * 278Glyext * 83 were enriched in the microsomal (MIC) fraction, similarly to the ER marker calnexin. PNS, post-nuclear supernatant. ( D ) In transfected HeLa cells, wild-type (wt) V5-DNAJB2a showed diffuse nuclear and cytoplasmic localization, whereas p. * 278Glyext * 83 (ext) partially colocalized with the endoplasmic reticulum (ER) visualized with the Cytopainter ER staining kit.

Article Snippet: Blots were stained with the following primary antibodies: calnexin Rb mAb C5C9 (Cell Signaling Technology, 2679, RRID:AB_2228381); DNAJB2 Rb pAb (Proteintech, 10 838-1-AP, RRID:AB_2277491); DNAJB6 Rb mAb [EPR17122] (Abcam, ab198995, RRID:AB_2924896); histone 3 Rb pAb (Abcam, ab1791, RRID:AB_302613); tubulin Rt mAb YL1/2 (Abcam, ab6160, RRID:AB_305328); V5 Ms mAb (Thermo Fisher, R960-25, RRID:AB_2556564); V5 Rb mAb D3H8Q (Cell Signaling Technology, 13202, RRID:AB_2687461).

Techniques: Membrane, Mutagenesis, In Silico, Sequencing, Stable Transfection, Transfection, Control, Extraction, Marker, Staining

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Turnover studies. ( A – B ) Wild-type (wt) or p. * 278Glyext * 83 (ext) DNAJB2a were expressed in T-REx 293 cells alone or in combination, and their levels were assayed at 0, 2 and 4 h of cycloheximide treatment. (A) A representative experiment performed in triplicate. 100 and 50 indicate a normalization sample at 100% and 50% loading, common for all three blots. (B) Quantification of three replicate experiments. The level of DNAJB2 was normalized to the transfection marker (GFP-V5) and represented relative to the initial level ( t = 0). Each data point represents the mean ± SD of one experiment performed in triplicate. Asterisks indicate significant differences in remaining protein amount at t = 4 compared with wild-type (2-tailed t -test; * P = 0.029, * * P = 0.001). DNAJB2 p. * 278Glyext * 83 showed an increased turnover rate and also increased the turnover of the co-expressed wild-type DNAJB2a. ( C – D ) T-REx 293 cells expressing DNAJB2 p. * 278Glyext * 83 were treated with cycloheximide alone (CH) or in combination with the proteasome inhibitor MG132 (MG) or lysosomal inhibitors (NH 4 Cl/leupeptin; NL) for 2 h. (D) The level of DNAJB2 was normalized to the transfection marker (GFP-V5) and represented relative to the initial level ( t = 0). The graph shows means ± SD from three replicate experiments, each performed in triplicate. MG132 efficiently blocked the turnover of mutant DNAJB2 ( * * P = 0.006, 2-tailed t -test).

Article Snippet: Blots were stained with the following primary antibodies: calnexin Rb mAb C5C9 (Cell Signaling Technology, 2679, RRID:AB_2228381); DNAJB2 Rb pAb (Proteintech, 10 838-1-AP, RRID:AB_2277491); DNAJB6 Rb mAb [EPR17122] (Abcam, ab198995, RRID:AB_2924896); histone 3 Rb pAb (Abcam, ab1791, RRID:AB_302613); tubulin Rt mAb YL1/2 (Abcam, ab6160, RRID:AB_305328); V5 Ms mAb (Thermo Fisher, R960-25, RRID:AB_2556564); V5 Rb mAb D3H8Q (Cell Signaling Technology, 13202, RRID:AB_2687461).

Techniques: Transfection, Marker, Expressing, Mutagenesis

Journal: Human Molecular Genetics

Article Title: Extension of the DNAJB2a isoform in a dominant neuromyopathy family

doi: 10.1093/hmg/ddad058

Figure Lengend Snippet: Oligomerization of DNAJB2. ( A ) Local sequence alignment of DNAJB2a (B2a) and DNAJB6b (B6b), with the coloured shadings indicating the J domain (JD), the glycine/phenylalanine-rich domain (G/F), the serine-rich region (SR) and the C-terminal domain (CTD) as defined for DNAJB6b by Karamanos et al . . Most of the region mediating DNAJB6b oligomerization (CTD β strands β1–β5) is highly similar between the two proteins. The C-terminal part of DNAJB2a (amino acids 218–277), not homologous to DNAJB6, is not shown. ( B – C ) Density gradient centrifugation of T-REx 293 cell lysates in 10–80% sucrose gradients. Fractionation profile of wild-type V5-DNAJB2a (a wt) suggests its oligomerization into polydisperse oligomers, similarly to endogenous DNAJB6 (a and b isoforms indicated) in the same samples (B). Co-expressed untagged wild-type (wt) and p. * 278Glyext * 83 (ext) DNAJB2a are both distributed throughout the gradient, albeit with somewhat different profiles (C). T, total samples.

Article Snippet: Blots were stained with the following primary antibodies: calnexin Rb mAb C5C9 (Cell Signaling Technology, 2679, RRID:AB_2228381); DNAJB2 Rb pAb (Proteintech, 10 838-1-AP, RRID:AB_2277491); DNAJB6 Rb mAb [EPR17122] (Abcam, ab198995, RRID:AB_2924896); histone 3 Rb pAb (Abcam, ab1791, RRID:AB_302613); tubulin Rt mAb YL1/2 (Abcam, ab6160, RRID:AB_305328); V5 Ms mAb (Thermo Fisher, R960-25, RRID:AB_2556564); V5 Rb mAb D3H8Q (Cell Signaling Technology, 13202, RRID:AB_2687461).

Techniques: Sequencing, Gradient Centrifugation, Fractionation