Journal: bioRxiv

Article Title: The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase

doi: 10.1101/2022.02.08.479504

Figure Lengend Snippet: (A-C) Cells expressing either Ssa1, 2, 3 or 4 as their sole Ssa and HA-tagged Rnr1/Rnr2/Rnr4 were grown to exponential phase and were either left untreated or were treated with 200 mM HU for 3 hrs. HA-RNR complexes were immunoprecipitated with anti-HA magnetic beads and were subjected to SDS-PAGE and analyzed by immunoblotting with anti-HA antibodies to detect the RNR subunits or anti-Ydj1 antibodies to detect Ydj1. (D) Interaction between RNR and Ydj1 (Ydj1/RNR) was calculated by quantitating bands from three replicate experiment. Each value represents the mean ± SD (n = 3). Statistical significance between samples was calculated ANOVA. (∗∗∗∗ p < 0.01).

Article Snippet: Proteins were detected using the following antibodies; anti-HA tag (Thermo #26183), Anti-FLAG tag (Sigma, #F1365), anti-PGK1 (Thermo # PA5-28612), anti-Ydj1 (StressMarq #SMC-166D).

Techniques: Expressing, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot

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