Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Abnormal NH 2 -terminal processing of the LGMDR1 mutants of CAPN3 within the PEF domain. A , schematic illustration of CAPN3 and its LGMDR1 mutants within the PEF domain. Stars indicate the cleavage sites recognized by the anti-AIS1 antibody. The bar indicates the antigen region (488–666 amino acids encoded by BC146672 ) of a commercial polyclonal anti-CAPN3 antibody (Proteintech, 28476-1-AP). . C129, H334, and N358 are catalytic centers. Arrows indicate autolytic cleavage sites. B , Western blotting of the cell lysates transfected with WT, protease-inactive form (C129S), and LGMDR1 mutants of EGFP-CAPN3 with anti-CAPN3 (28476-1-AP, upper panel ) and anti-AIS1 ( lower panel ) antibodies. The cell lysates of CAPN3:CS lack the two AIS1-recognizing fragments. C , band intensity ratio of “fragment b” to “fragment a” in the LGMDR1 mutants within PEF domains. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. AIS1, autolysis within IS1; NS, N-terminal sequence; IS, internal sequence; PC, protease core; CBSW, calpain-type β-sandwich; PEF, penta-EF motif; LGMDR1, limb-girdle muscular dystrophy R1; CAPN, calpain.

Article Snippet: We then examined their autolytic processing (a hallmark of CAPN3 activation) patterns using a commercially available anti-CAPN3 antibody (Proteintech, 28476-1-AP) and our recently developed anti-AIS1 (autolysis within IS1) antibody, which specifically detects the IS1 autolytic cleavage site of CAPN3 ( ).

Techniques: Western Blot, Transfection, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Oligomerization of LGMDR1 mutants of CAPN3 within the PEF domain. A , Western blotting of LGMDR1 mutant forms of EGFP-CAPN3 with ( right panel ) or without ( left panel ) cross-linking by 0.1% glutaraldehyde. B , ratio of the band intensity of oligomer (>250 kDa) to monomer (∼130 kDa) forms of the LGMDR1 mutants within the PEF domain. ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. C , 3D structural model of the CAPN3 dimer and position of the I807 amino acid residue ( red ) within the EF5 loop (Protein Data Bank ID: 4OKH ) . D , oligomerization of CAPN3:CS and CAPN3:CS:I807T expressed in HEK293T cells. E , relative ratio of oligomer to monomer. ∗ p = 0.0121, Mann–Whitney U test. F , Western blotting of the cell lysates expressing WT and the I807T mutant form of EGFP-CAPN3 with anti-CAPN3 ( upper panel ) and anti-AIS1 ( lower panel ) antibodies. G , band intensity ratio of “fragment b” to “fragment a” of EGFP-CAPN3:I807T. ∗∗∗ p = 0.00084, Mann–Whitney U test. H , scatter plot of the oligomer/monomer ratio against the NH 2 -terminal processing ratio (b/a) of various LGMDR1 mutants within the PEF domain. Mutants in red lack “fragment b” when expressed in HEK293T cells. I807T in light blue is an artificial mutant that lacks “fragment b” in the cell lysates. PEF, penta-EF motif; LGMDR1, limb-girdle muscular dystrophy R1; AIS1, autolysis within internal sequence1; CAPN, calpain; EGFP, enhanced green fluorescent protein.

Article Snippet: We then examined their autolytic processing (a hallmark of CAPN3 activation) patterns using a commercially available anti-CAPN3 antibody (Proteintech, 28476-1-AP) and our recently developed anti-AIS1 (autolysis within IS1) antibody, which specifically detects the IS1 autolytic cleavage site of CAPN3 ( ).

Techniques: Western Blot, Mutagenesis, Comparison, Residue, MANN-WHITNEY, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Analysis of CAPN3 binding to the two titin regions in cells with heterologous overexpression. A , schematic illustration of the mCherry-fusion proteins with the two regions of human titin (NCBI reference sequence: NP_596869.4 ). The N2A region corresponds to the mouse mCN48 fragment identified as a p94-binding fragment via yeast two-hybrid screening in our previous study . B , EGFP-CAPN3:CS is coimmunoprecipitated with mCherry-titin CT more efficiently than with mCherry-titin I80-PEVK in HEK293T cells. Because of their weak interaction, the cells were treated with a dithiobis(succinimidyl propionate) cross-linker before cell lysis and immunoprecipitation. C , band intensity ratio of the coimmunoprecipitated CAPN3 to the CAPN3 in the lysate. ∗ p = 0.0211, Mann–Whitney U test . D , immunostaining of EGFP-CAPN3:CS ( green ) coexpressed with mCherry fusion proteins of the two titin regions ( magenta ) in HeLa cells. Arrows ( upper panel ) and arrowheads ( bottom panel ) show filopodia with no GFP staining. The scale bar represents 20 μm. E , translocation of EGFP-CAPN3:CS to the membrane fractions by mCherry-titin CT-kRas, but not mCherry-titin I80-PEVK-kRas, in HeLa cells. HeLa cells were fractionated into cytosol and membrane fractions. The upper and the lower panels show the expression of EGFP-CAPN3:CS (Blot: anti-CAPN3 antibody), and mCherry-titin-kRas (Blot: anti-RFP antibody), respectively, and the protein bands were labeled with arrows . F , ratio of band intensity of EGFP-CAPN3:CS in the membrane fraction to that in the cytosol fraction. ∗∗ p = 0.0073, one-way ANOVA with Dunnett’s multiple comparison test. a.a., amino acid number; LGMDR1, limb-girdle muscular dystrophy R1; CAPN, calpain; EGFP, enhanced green fluorescent protein; CT, COOH terminal; RFP, red fluorescent protein.

Article Snippet: We then examined their autolytic processing (a hallmark of CAPN3 activation) patterns using a commercially available anti-CAPN3 antibody (Proteintech, 28476-1-AP) and our recently developed anti-AIS1 (autolysis within IS1) antibody, which specifically detects the IS1 autolytic cleavage site of CAPN3 ( ).

Techniques: Binding Assay, Over Expression, Sequencing, Two Hybrid Screening, Lysis, Immunoprecipitation, MANN-WHITNEY, Immunostaining, Staining, Translocation Assay, Membrane, Expressing, Labeling, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Localization of LGMDR1 mutants of EGFP-CAPN3 and mCherry-titin CT in HeLa cells. A , immunostaining of EGFP-CAPN3:CS ( green ) and its LGMDR1 mutants coexpressed with mCherry-titin CT ( magenta ) in HeLa cells. The scale bar represents 10 μm. B , quantification of the colocalization of EGFP-CAPN3:CS and its LGMDR1 mutants with mCherry-titin CT. Pearson correlation coefficient was calculated using Coloc2 software. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. LGMDR1, limb-girdle muscular dystrophy R1; CAPN, calpain; EGFP, enhanced green fluorescent protein; CT, COOH terminal.

Article Snippet: We then examined their autolytic processing (a hallmark of CAPN3 activation) patterns using a commercially available anti-CAPN3 antibody (Proteintech, 28476-1-AP) and our recently developed anti-AIS1 (autolysis within IS1) antibody, which specifically detects the IS1 autolytic cleavage site of CAPN3 ( ).

Techniques: Immunostaining, Software, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Localization of EGFP-CAPN3 in TA muscles of Capn3 −/− mice following AAV-mediated expression. A , immunohistochemistry of AAV-infected EGFP, EGFP-CAPN3, EGFP-CAPN3:CS, EGFP-CAPN3:CS:DIS2 (GFP, green ), and actinin ( magenta ) in Capn3 −/− mice ( left panels ). Right panels show intensity plots of EGFP and actinin immunoreactivity along the line in each merged image. The scale bar represents 10 μm. B , immunohistochemistry of endogenous CAPN3 and actinin in the gastrocnemius muscle of 12-week-old Capn3 +/+ and Capn3 −/− mice. Tissues were stained with anti-CAPN3 ( green , Proteintech 28476-1-AP) and anti-actinin ( magenta ) antibodies ( left panels ). Right panels indicate the line plot of CAPN3 and actinin immunosignals within the merged images. The scale bar represents 10 μm. CAPN, calpain; EGFP, enhanced green fluorescent protein; AAV, adeno-associated virus; CT, COOH terminal; TA, tibialis anterior.

Article Snippet: We then examined their autolytic processing (a hallmark of CAPN3 activation) patterns using a commercially available anti-CAPN3 antibody (Proteintech, 28476-1-AP) and our recently developed anti-AIS1 (autolysis within IS1) antibody, which specifically detects the IS1 autolytic cleavage site of CAPN3 ( ).

Techniques: Muscles, Expressing, Immunohistochemistry, Infection, Staining, Virus

Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Subcellular localization of LGMDR1 mutants of EGFP-CAPN3:CS in Capn3 −/− mice TA following AAV-mediated expression. A–C , immunohistochemistry of the AAV-infected LGMDR1 mutant form of EGFP-CAPN3:CS (anti-GFP antibody, green ) and anti-actinin ( magenta ) antibody ( left panels ) in Capn3 −/− mice. Right panels show the intensity plot of EGFP and actinin immunoreactivity along the line in each merged image. Three patterns of CAPN3 immunosignals were observed in the LGMDR1 mutants within the PEF domain: fine M-band signals ( A ), broadened M-band signals ( B ), and signals around the N2A region of sarcomeres ( C ). The scale bar represents 10 μm. LGMDR1, limb-girdle muscular dystrophy R1; CAPN, calpain; EGFP, enhanced green fluorescent protein; AAV, adeno-associated virus; TA, tibialis anterior; PEF, penta-EF hand.

Article Snippet: We then examined their autolytic processing (a hallmark of CAPN3 activation) patterns using a commercially available anti-CAPN3 antibody (Proteintech, 28476-1-AP) and our recently developed anti-AIS1 (autolysis within IS1) antibody, which specifically detects the IS1 autolytic cleavage site of CAPN3 ( ).

Techniques: Expressing, Immunohistochemistry, Infection, Mutagenesis, Virus

Journal: The Journal of Biological Chemistry

Article Title: Oligomer-dependent and oligomer-independent pathogenesis of muscular dystrophy-associated mutations within the penta-EF-hand domain of calpain-3

doi: 10.1016/j.jbc.2026.111277

Figure Lengend Snippet: Partial rescues of abnormal intracellular localization of EGFP-CAPN3:CS:A702V but not of EGFP-CAPN3:CS:D705H in Capn3 +/+ mice. A , immunostaining of actinin ( magenta ) and EGFP-CAPN3:CS:A702V and EGFP-CAPN3:CS:D705H ( green ) in the TA muscles of Capn3 +/+ mice. Right panel shows the line plot of the merged image. The scale bar represents 10 μm. B , coexpression of CAPN3:CS ameliorated the localization of EGFP-CAPN3:CS:A702V but not EGFP-CAPN3:CS:D705H at the mCherry-titin CT ( magenta ) enriched plasma membrane in HeLa cells. Right panels show the line plot of mCherry-titin CT ( magenta ) and EGFP-CAPN3:CS, CAPN3:CS:A702V, and CAPN3:CS:D705H ( green ) within the left merged images. C , quantification of the colocalization of EGFP-CAPN3:CS and its LGMDR1 mutants with mCherry-titin CT upon coexpression of CAPN3 in HeLa cells. Pearson correlation coefficient was calculated using Coloc2 software. ∗∗∗ p = 0.0019, one-way ANOVA with Bonferroni multiple comparison test. LGMDR1, limb-girdle muscular dystrophy R1; CT, COOH terminal; CAPN, calpain; EGFP, enhanced green fluorescent protein; TA, tibialis anterior.

Article Snippet: We then examined their autolytic processing (a hallmark of CAPN3 activation) patterns using a commercially available anti-CAPN3 antibody (Proteintech, 28476-1-AP) and our recently developed anti-AIS1 (autolysis within IS1) antibody, which specifically detects the IS1 autolytic cleavage site of CAPN3 ( ).

Techniques: Immunostaining, Muscles, Clinical Proteomics, Membrane, Software, Comparison

Journal: Scientific Reports

Article Title: Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation

doi: 10.1038/srep13474

Figure Lengend Snippet: ( A ) Phosphorylated LIX1L was immunoprecipitated from the cytosolic and nuclear fractions of HEK-293FLG and HEK-293FLG-LIX1L cells using a FLAG antibody. Immunoprecipitates were analyzed through western blot analysis with a LIX1L antibody and phosphorylated serine-, threonine- and tyrosine-specific antibodies (upper panels). In the cytosolic and nuclear fractions of the HEK-293FLG and HEK-293FLG-LIX1L cells treated with 25 μM PY95 as a negative control or PY136, immunoprecipitates obtained using the FLAG antibody were analyzed through a western blot analysis with the LIX1L antibody and phosphorylated tyrosine-specific antibodies (bottom panels). Representative blots from HEK-293FLG and HEK-293FLG-LIX1L cell lines are shown. ( B ) The cell counts of HEK-293FLG and HEK-293FLG-LIX1L cells after treatment with PY136 (left panel). The HEK-293FLG and HEK-293FLG-LIX1L cells were cultured in semisolid methylcellulose media. The HEK-293FLG-LIX1L cells were left untreated or were treated with PY136 (25 μM). After 14 days in culture, colony formation was analyzed, and the cells were viewed using phase-contrast microscopy. The colonies formed from each cell type (3 × 10 2 to 5 × 10 2 cells/plate) were counted following plating onto semisolid methylcellulose media (right upper panel). Original magnification 4x (right bottom panels). These data are shown as the mean ± SD for independent experiments. **p < 0.05, *p < 0.01 . ( C ) The results of the immunoblot analysis of the cytosolic fraction treated with or without RNase in HEK-293FLG-LIX1L cells. The black arrow indicates the FLAG-LIX1L fusion protein. The red arrows indicate the detected proteins associated with the LIX1L-RNA complex. ( D ) Western blot analysis revealed that LIX1L interacted with the RIOK1, nucleolin and PABPC4 proteins in the cytoplasm of HEK-293 cells. In ( A ) and ( D ), the cropped blots were run under the same experimental condition.

Article Snippet: A Rabbit anti-LIX1L polyclonal antibody (Abnova, Taipei, Taiwan), mouse anti-FLAG monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phosphothreonine polyclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-phosphoserine polyclonal antibody (Abcam), rabbit anti-phosphotyrosine polyclonal antibody (Abcam), rabbit anti-RIOK1 polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-nucleolin polyclonal antibody (Origene, Rockville, MD, USA), rabbit anti-PABPC4 polyclonal antibody (Abnova), rabbit anti-Cofilin polyclonal antibody (Cell Signaling Technology (CST), Beverly, MA, USA), rabbit anti-phospho-Cofilin (Ser 3) polyclonal antibody (CST), rabbit anti-α Tubulin polyclonal antibody (Abcam), rabbit anti-Lamin A + C monoclonal antibody (Abcam), rabbit anti-ROS1 monoclonal antibody (CST) and anti-Actin antibody (Abcam) were used in the present study.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Cell Culture, Microscopy

Journal: Scientific Reports

Article Title: Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation

doi: 10.1038/srep13474

Figure Lengend Snippet: The identification of LIX1L-associated proteins by MALDI-TOF/TOF mass spectrometry.

Article Snippet: A Rabbit anti-LIX1L polyclonal antibody (Abnova, Taipei, Taiwan), mouse anti-FLAG monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phosphothreonine polyclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-phosphoserine polyclonal antibody (Abcam), rabbit anti-phosphotyrosine polyclonal antibody (Abcam), rabbit anti-RIOK1 polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-nucleolin polyclonal antibody (Origene, Rockville, MD, USA), rabbit anti-PABPC4 polyclonal antibody (Abnova), rabbit anti-Cofilin polyclonal antibody (Cell Signaling Technology (CST), Beverly, MA, USA), rabbit anti-phospho-Cofilin (Ser 3) polyclonal antibody (CST), rabbit anti-α Tubulin polyclonal antibody (Abcam), rabbit anti-Lamin A + C monoclonal antibody (Abcam), rabbit anti-ROS1 monoclonal antibody (CST) and anti-Actin antibody (Abcam) were used in the present study.

Techniques: Virus

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 1. Cytoplasmic form of nucleolin. (A) The N-terminal, middle domain, and C-terminal immunogens used to raise nucleolin antibodies. (B) Nucleolin immunoblot of cytoplasmic and nuclear fractions by antibodies raised against the N-terminal, middle domain, and C-terminal immunogens. All antibodies detect a single band with a MW ≅110 kDa in SDS-PAGE. (C) Actin, HDAC1, and Pan3 immunoblots show purity of cytoplasmic and nuclear lysates of Hey ovarian cancer cells. (D) Nucleolin immunoblot of cytoplasmic and nuclear fractions detects a single band with a pI ≅4.6 in IEF. (E) Subcellular localization of nucleolin by indirect immunofluorescence microscopy in MCF10A non-cancerous epithelial breast cells. The N-terminal raised antibody does not detect nucleolin in either the cytoplasm or nucleus. The middle domain raised antibody detects nucleolin both in the cytoplasm and nucleus. The C-terminal raised antibody detects nucleolin in the nucleus only. (F) Subcellular localization of nucleolin in MDA-MB-231 breast cancer cells. The N-terminal raised antibody detects nucleolin in the cytoplasm only. The middle domain raised antibody detects nucleolin both in the cytoplasm and nucleus. The C-terminal raised antibody detects nucleolin only in the nucleus, primarily in the nucleolus. Nucleolin shRNA decreased nucleolin signal in the cytoplasm detected by the N-terminal raised antibody. (G) The cytoplasmic form of nucleolin in MDA-MB-231 cells detected by the N-terminal and middle domain raised antibodies is co-localized. The bar is 10 μm.

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques: Western Blot, SDS Page, Microscopy, shRNA

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 3. Nucleolin interacts with the hl structure in the 5′UTR as well as G-quadruplex in the 3′UTR of CSF-1 mRNA. (A) RNA electrophoretic mobility shift assay shows an association of nucleolin (tagged with myc-DDK) with the hl structure from the 5′UTR as well as with the G-quadruplex from the 3′UTR of CSF-1 mRNA. G-quadruplex is used as a nucleolin binding control [17]. Nucleolin does not associate with either the linear 5′hl sequence or 5′UTR without hl sequence. (B) Luciferase protein level is increased (4– 5-fold) in wild type construct (Wt = Wt) in response to the nucleolin overexpression in SKOV3 cells. In contrast, luciferase protein level is not increased in the hl deletion construct {D(21) = Wt} in response to the nucleolin overexpression. (C) Luciferase mRNA stability is decreased in D(21) = Wt (red solid line) compared to Wt = Wt (black solid line) in SKOV3 cells. However, there was no significant effect of nucleolin overexpression on luciferase mRNA stability (broken lines). The cDNA was synthesized by random primer (pdN6). This suggests an effect of nucleolin on luciferase mRNA translation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Control, Sequencing, Luciferase, Construct, Over Expression, Synthesized

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 4. Nucleolin forms mRNP complex with eIF4G and CSF-1 mRNA in Hey ovarian cancer cells. (A) N-terminal raised antibody (N-NCL) immunoprecipitates nucleolin only in RNases- treated sample. In the same sample, nucleolin co-immunoprecipitates with eIF4G. (B) Middle domain raised antibody (M-NCL) immunoprecipitates nucleolin in all treatments. However, nucleolin did not co-immunoprecipitate with eIF4G in the same samples. (C) C-terminal raised antibody (C-NCL) immunoprecipitates nucleolin. However, nucleolin did not co-immunoprecipitate with eIF4G in the same samples. (D) eIF4G antibody immunoprecipitates eIF4G in all treatments. However, eIF4G co-immunoprecipitates with nucleolin only in RNases-treated sample. (E) RT-qPCR measurements of CSF-1 mRNA in nucleolin or eIF4G immunoprecipitates of Hey cell lysates show direct interaction between either nucleolin or eIF4G and CSF-1 mRNA. After IP of RNA-protein complexes from Hey cell lysates, RNA was isolated and used in RT reactions and amplified by real time PCR. The graph shows relative mRNA levels in either nucleolin or eIF4G IP compared with control IgG IP conditions. The mean ± SD of CSF-1 mRNA normalized for GAPDH mRNA is depicted (n = 3). GAPDH mRNA was set to equal 1.

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques: Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 5. Cytoplasmic nucleolin co-localizes with the eIF4G in MDA-MB-231 breast cancer cells. The cytoplasmic nucleolin (green) detected by the N-terminal raised antibody co-localized with eIF4G (red). The bar is 10 μm.

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques:

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 7. Formation of a nucleolin-mediated mRNP in translational regulation. Binding of nucleolin to the hl structure in the 5′UTR attracts eIF4F complex formation and enhances translation. Binding of nucleolin to the 3′UTR facilitates miRISC formation resulting in miRNA-directed deadenylation, but blocks further mRNA decay.

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Dependency Map correlation analysis reveals WDR89 as a genome maintenance factor

doi: 10.64898/2026.01.28.698016

Figure Lengend Snippet: WDR89 is a nucleolar protein that responds to double-strand DNA break-inducing stresses. ATM-dependent nucleolar accumulation of WDR89 upon genotoxic stress. (A) WDR89 localizes predominantly in the nucleus and overlaps largely with nucleolin. (B) Quantification of WDR89 nuclear staining intensity following ionizing radiation. (C) Quantification of WDR89 nuclear staining intensity following genotoxic chemical treatments. (D) Confirmation of DNA damage by γH2AX foci staining of the conditions in C. (E) Effect of ATM inhibitor treatment on WDR89 nuclear intensity changes after IR. (F) Western blot of chromatin fractionation for WDR89 and the indicated markers. For panels in B, C, D and E, ****p<0.0001, by ANOVA with Tukey’s post-hoc test or by T-test where appropriate.

Article Snippet: Immunofluorescence staining was performed using anti-γ-H2AX antibodies (Abcam, ab81299, Santa Cruz, sc-517348), an anti-Nucleolin antibody (Novus, NBP2-44610), anti WDR89 antibody (Bethyl, A301-872A) or p53 antibody (Cell Signaling, #9282) at 1:250 dilution and Alexafluor 568 goat anti-rabbit secondary and Alexafluor 488 goat anti-mouse antibodies (Invitrogen) at 1:400 dilution.

Techniques: Staining, Western Blot, Fractionation

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Schematic diagrams of α-syn and DNAJC5. Domains are highlighted in different colors. Red arrows indicate known disease-causing mutations on each protein. ( B ) Membrane and cytosol fractionation scheme. Briefly, homogenized HEK293T cells were centrifuged at low speed to prepare a post-nuclear supernatant (PNS). High-speed centrifugation was then performed to separate the sedimentable membrane (M) from cytosol (C). ( C ) Partition of palmitoylated DNAJC5 (P-DNAJC5) and non-palmitoylated DNAJC5 (NP-DNAJC5) between the membrane (M) and cytosol (C) fractions. DNAJC5 was immunoprecipitated from cytosol and membrane with anti-FLAG resin and evaluated by Coomassie-blue stained SDS-PAGE. ( D ) α-syn secretion 16 h after transfection. The secretion of P-DNAJC5 in the medium was detected. ( E ) α-syn secretion 36 h after transfection. NP-DNAJC5 was also secreted in the medium together with α-syn. Figure 1—source data 1. Uncropped immunoblot and gel images corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Membrane, Fractionation, Centrifugation, Immunoprecipitation, Staining, SDS Page, Transfection, Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Membrane and cytosol fractionation of various cell lines (HEK293T, MDA-MB-231, and Hela) transfected with DNAJC5. The fractionation was performed as depicted in . C, cytosol; M, membrane; PNS, post-nuclear supernatant. Transferrin receptor (TFR) was used as a membrane marker. Tubulin was used as a cytosol marker. ( B ) In vitro depalmitoylation assay. Sedimented membranes (M) from different DNAJC5-transfected cell lines were collected and resuspended in 0.25 M Tris pH 7.2 buffer or 0.25 M Hydroxylamine (HA) pH 7.2 buffer. After overnight incubation at room temperature, the samples were examined by SDS-PAGE followed by anti-DNAJC5 immunoblot. PNS and C were used to compare the mobility of P-DNAJC5 and NP-DNAJC5, respectively. Figure 1—figure supplement 1—source data 1. Uncropped immunoblot images corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Membrane, Fractionation, Transfection, Marker, In Vitro, Incubation, SDS Page, Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Trypan blue cell vital staining after transfection with various constructs used in . Ratios of trypan blue positive cells indicate the toxicity caused by transfection. Error bars represent standard deviations of three samples. ( B ) Secretion of α-syn variants into conditioned medium. Medium was collected, concentrated, and evaluated by SDS-PAGE and immunoblot. ( C ) Expression of α-syn variants in HEK293T cells. HEK293T cells were co-transfected with Parkinson’s disease (PD)-causing α-syn mutant (A30P, E46K, and A53T) and DNAJC5. ( D ) Quantification of normalized secretion (amount in medium divided by amount in lysate) of various α-syn variants and DNAJC5. The quantification was based on immunoblot in ( B ) and ( C ). Figure 1—figure supplement 2—source data 1. Uncropped immunoblot images corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Staining, Transfection, Construct, SDS Page, Western Blot, Expressing, Mutagenesis

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Schematic diagram of nanoluciferase (Nluc)-fused α-syn. ( B ) Nluc-α-syn secretion was stimulated by DNAJC5 expression. Plasmids encoding Nluc-α-syn and DNAJC5 were co-transfected into HEK293T cells. Expression and secretion of proteins were detected with immunoblot 36 hr after transfection. ( C ) Time-dependent of accumulation of Nluc-α-syn in the medium without DNAJC5 overexpression. After transfecting HEK293T cells with Nluc-α-syn alone, fractions of medium were collected at indicated time points. ( D ) Chemical structure of quercetin (QLT), a reported DNAJC5 inhibitor. ( E ) QLT inhibited endogenous Nluc-α-syn secretion in a concentration-dependent manner. AU, arbitrary unit. Secretion assay similar to ( B ) was performed with treatment of indicated concentration of QLT. Amounts of secreted proteins were quantified with nanoluciferase assay 36 hr after transfection. Immunoblot of α-syn and DNAJC5 in cell lysate after QLT treatment was shown on the right. Error bars represent standard deviations of three samples. ( F ) Validation of DNAJC5 knockout (KO) cell line generated by CRISPR. Wildtype (WT) HEK293T cell was used as a control. In all three single clones of DNAJC5 KO cell lines, DNAJC5 was not detectable by immunoblot. ( G ) Treatment of bafilomycin A1 (BaFA1) induced LC3 lipidation in cells. After 24 hr of 100 nM BaFA1 treatment, media were collected, and cells were lysed for evaluation with SDS-PAGE followed by immunoblot. The accumulation of the lipidated form of LC3 (LC3-II) was used to indicate the inhibition of autophagy and lysosomal degradation in cells. ( H ) Quantification of α-syn secretion from WT and DNAJC5 KO HEK293T cells after 24 hr treatment of BaFA1. The normalized secretion was calculated as nanoluciferase reading from media divided by the reading from cell lysate. Error bars represent standard deviations of three samples. Figure 1—figure supplement 3—source data 1. Uncropped immunoblot images corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Expressing, Transfection, Western Blot, Over Expression, Concentration Assay, Biomarker Discovery, Knock-Out, Generated, CRISPR, Control, Clone Assay, SDS Page, Inhibition

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Medium fractionation scheme. ( B ) Secreted α-syn was soluble. Differential centrifugation was performed with conditioned medium from HEK293T cells transfected with DNAJC5 and α-syn. Alix and CD9, exosome markers. PDI, an endoplasmic reticulum (ER) marker, was used as exosome-negative control. ( C ) Gel filtration fractionation of medium. Conditioned medium was concentrated and subjected to gel filtration fractionation. Fractions were evaluated by anti-α-syn immunoblot. ( D ) Chromatograms of tandem α-syn monomer (blue curve), dimer (green curve), and tetramer (red curve) were overlaid. In comparison, the relative intensity of secreted α-syn in each fraction was plotted as blue bars. ( E ) Schematic diagram of co-immunoprecipitation (co-IP) of secreted α-syn and FLAG-α-syn. Shown here is possible interaction between α-syn (blue circle) and FLAG-α-syn (yellow circle) in a representative tetrameric conformation. ( F ) Anti-FLAG immunoprecipitation (FLAG-IP) of media from cells transfected with indicated plasmids. Both the medium input and FLAG-IP samples were evaluated with anti-α-syn immunoblot (anti-α-syn WB). Figure 2—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Fractionation, Centrifugation, Transfection, Marker, Negative Control, Filtration, Western Blot, Comparison, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Medium fractionation of secreted α-syn PD mutants (A30P, E46K, and A53T). After differential centrifugation of medium, supernatant (10ks and 100ks) and pellet fractions (100kp) were evaluated by immunoblot. ( B ) Medium fractionation of basal secreted Nluc-α-syn without DNAJC5 overexpression. Similar fractionation assay was performed in ( A ) with medium from HEK293T cells transfected with Nluc-α-syn alone. Secreted Nluc-α-syn was quantified with a nanoluciferase assay. AU, arbitrary unit. Figure 2—figure supplement 1—source data 1. Uncropped immunoblot images corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Fractionation, Centrifugation, Western Blot, Over Expression, Transfection

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Schematic diagram of EV flotation protocol. Briefly, high-speed pellet fractions of growth medium were resuspended in 60% sucrose buffer and overlaid sequentially with 40% and 10% sucrose buffer. The tubes were centrifuged at 150,000 (150k)× g at 4°C for overnight. Buoyant EVs floated at the 10%/40% sucrose interface, separated from other insoluble materials. ( B ) Immunoblots across the sucrose step gradient. DNAJC5 as well as other classical exosome markers (Flot-2, CD63, and CD9) were highly enriched in fractions 4–6 at the 10%/40% interface. Figure 2—figure supplement 2—source data 1. Uncropped immunoblot images corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Inhibition of DNAJC5 palmitoylation by 2-bromopalmitic acid (2-BA) or introduced mutation L115R. Cellular fractionation was performed with HEK293T cells transfected with WT DNAJC5 and treated with 10 μm 2-BA, or transfected with DNAJC5 L115R mutant. C, cytosol; M, membrane; PNS, post-nuclear supernatant; TFR, transferrin receptor. ( B ) Quantification of the percentage of P-DNAJC5 and NP-DNAJC5 in different conditions as shown in ( A ). Error bars represent standard deviations of three experiments. ( C ) α-syn secretion was blocked with 2-BA treatment. HEK293T cells transfected with indicated plasmids were treated with DMSO or 10 μm 2-BA. Media fractions were collected and secretion was evaluated by SDS-PAGE and immunoblot. ( D ) Palmitoylation of DNAJC5 was blocked in HEK293T cells treated with 2-BA. ( E ) Quantification of normalized α-syn secretion in HEK293T cells after 2-BA treatment. The quantification was based on immunoblot in ( C ) and ( D ). The α-syn secretion was calculated as the amount of α-syn in media divided by the amount in lysate. ( F ) Quantification of normalized DNAJC5 secretion in HEK293T cells after 2-BA treatment. The quantification was based on immunoblot in ( C ) and ( D ). The DNAJC5 secretion was calculated as the amount of DNAJC5 in media divided by the amount in lysate. ( G ) DNAJC5 L115R mutant reduced α-syn secretion compared with WT DNAJC5. Secretion assay with HEK293T cells transfected with indicated plasmids encoding DNAJC5 variant was performed similar to ( C ). ( H ) DNAJC5 L115R was non-palmitoylated in HEK293T cells. ( I ) Quantification of normalized α-syn secretion in HEK293T cells transfected with DNAJC5 L115R mutant. The quantification was based on immunoblot in ( G ) and ( H ). ( J ) Quantification of normalized DNAJC5 secretion in HEK293T cells transfected with DNAJC5 L115R mutant. The quantification was based on immunoblot in ( G ) and ( H ). Figure 3—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Inhibition, Mutagenesis, Cell Fractionation, Transfection, Membrane, SDS Page, Western Blot, Variant Assay

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Chemical structure of palmitoylation inhibitor 2-bromopalmitic acid (2-BA). The single bromo substituent at position 2 is highlighted by a red dashed square. ( B ) α-syn secretion in the medium was inhibited by 2-BA in a dose-dependent manner. About 10 μm 2-BA was serially diluted by DMSO into 5 μm, 2 μm, and 1 μm solution. HEK293T cells were first transfected with DNAJC5 and α-syn. After medium replacement, 2-BA of indicated concentration was added to cell culture. Media were collected after 36 hr and followed by sample preparation, SDS-PAGE and immunoblot. ( C ) With increasing concentration of 2-BA, P-DNAJC5 decreased and NP-DNAJC5 increased in HEK293T cells. ( D ) Quantification of normalized α-syn secretion upon increasing concentration of 2-BA. Quantification was based on immunoblot in ( B ) and ( C ). The α-syn secretion was calculated as the amount of α-syn in media divided by the amount in lysate. ( E ) Quantification of ratio of P-DNAJC5/NP-DNAJC5. The quantification was based on immunoblot in ( C ). Figure 3—figure supplement 1—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Transfection, Concentration Assay, Cell Culture, Sample Prep, SDS Page, Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) USP19 induced α-syn secretion in the medium, which was further enhanced by WT DNAJC5 and blocked by two DNAJC5 palmitoylation-deficient mutants (L115R and L116Δ). ( B ) Both mutations, L115R and L115Δ, inhibited DNAJC5 palmitoylation in HEK293T cells. ( C ) Quantification of normalized α-syn secretion in HEK293T cell transfected with indicated constructs. The quantification was based on immunoblot in ( A ) and ( B ). α-Syn secretion was calculated as the amount of α-syn in media divided by the amount in lysate. Figure 3—figure supplement 2—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Transfection, Construct, Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Immunofluorescence (IF) images of U2OS cells transfected with wildtype (WT) DNAJC5-HaloTag. Before fixation, HaloTag TMR ligands were added to the cell culture to label DNAJC5. Golgi apparatus was visualized by IF using anti-GM130 antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. ( B ) IF images of U2OS cells transfected with DNAJC5 (L115R)-HaloTag. Same staining procedure was performed as in ( A ). Scale bar: 20 μm. ( C ) IF images of U2OS cells transfected with WT DNAJC5-HaloTag and treated with 5 μM 2-BA. After transfection with WT DNAJC5-HaloTag, cells were incubated with 5 μM 2-BA for 1 day to block palmitoylation of DNAJC5. Same staining procedure was performed as in ( A ). Scale bar: 10 μm.

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Immunofluorescence, Transfection, Cell Culture, Staining, Incubation, Blocking Assay

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Colocalization between endogenous CD63 (red) and DNAJC5 (green). U2OS cells were cultured and fixed, incubated with corresponding antibodies for IF detection of endogenous CD63 and DNAJC5. Representative colocalized region is shown in magnified insets. Scale bar: 20 μm. ( B ) CD63 (green) was inside the enlarged endosomes labeled by peripheral mCherry-Rab5 Q79L (red). U2OS expressing mCherry-Rab5 Q79L were fixed, followed by IF using anti-CD63 antibody. Scale bar: 25 μm.

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Cell Culture, Incubation, Labeling, Expressing

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) DNAJC5 was internalized inside enlarged endosomes. Live U2OS cells expressing mCherry-Rab5 Q79L (red) showed circular enlarged endosomes labeled by Rab5 mutant. DNAJC5-HaloTag (green) was visualized by addition of HaloTag Oregon Green Ligand. Representative enlarged endosomes show diffuse (1) or punctate (2 and 3) internalized DNAJC5. Scale bar: 15 μm in overviews and 1 μm in magnified insets. ( B ) α-syn was excluded from enlarged endosomes. In live U2OS cells, expression of BFP-Rab5 Q79L (blue) produced enlarged endosomes of similar morphology compared with mCherry-Rab5 Q79L . mNeonGreen-α-syn (mNG-α-syn, green) was expressed both in the nucleus and cytosol. No mNG-α-syn was found inside enlarged endosomes (1–3). Scale bar: 20 μm in overviews and 1 μm in magnified insets. ( C ) α-syn enters into enlarged endosomes in the presence of DNAJC5. DNAJC5-HaloTag (red) and mNG-α-syn (green) were coexpressed in U2OS cells carrying BFP-Rab5 Q79L (blue) mutant and imaged. No mNG-α-syn was internalized in endosome without DNAJC5-HaloTag inside (1). In contrast, mNG-α-syn was found inside endosomes with DNAJC5-HaloTag inside (2 and 3). Scale bar: 10 μm in overviews and 1 μm in magnified insets. ( D ) α-syn and DNAJC5 co-sedimented in membrane fractionation. HEK293T cell homogenate was sequentially centrifuged at increasing velocity from 3000× g (3k), 25,000× g (25k), and 100,000× g (100k). The 25k membrane fraction had the highest amount of both α-syn and DNAJC5. ( E ) Quantification of the membrane fractionation results in ( D ). ( F ) Proteinase K protection assay of 25k membrane-containing α-syn and DNAJC5. ( G ) Quantification of the proteinase K protection assay in ( F ). ( H ) Quantification of the ratio of α-syn-containing endosomes in control cells (no-DNAJC5 transfection) or cells co-transfected with DNAJC5. More than 100 enlarged endosomes were counted in each group. Error bars represent standard deviations. P value<0.0001, two-tailed t test. Figure 4—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Expressing, Labeling, Mutagenesis, Produced, Membrane, Fractionation, Control, Transfection, Two Tailed Test, Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Live-cell imaging of U2OS cells transfected with DNAJC5 (L115R)-HaloTag and mCherry-Rab5 Q79L . DNAJC5 (L115R)-HaloTag (green) is diffuse in cytosol and outside of the enlarged endosomes labeled by peripheral mCherry-Rab5 Q79L (red). Scale bar: 10 μm in overviews and 1 μm in magnified insets. ( B ) Live-cell imaging of U2OS cells transfected with mNeonGreen (mNG)-α-syn, DNAJC5 (L115R)-HaloTag, and mCherry-Rab5 Q79L . In the condition of coexpression with DNAJC5 (L115R)-HaloTag (red), no mNG-α-syn (green) was found inside of enlarged endosomes labeled by peripheral BFP-Rab5 Q79L (blue). Scale bar: 10 μm in overviews and 1 μm in magnified insets.

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Live Cell Imaging, Transfection, Labeling

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: Medium fractionation of secreted tandem α-syn oligomers. P-DNAJC5 was depleted in the supernatant after centrifugation at 100,000 (100k)× g . However, no significant decrease of tandem α-syn oligomers in the supernatant was observed. Figure 5—figure supplement 1—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Fractionation, Centrifugation, Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Ladder pattern of higher molecular weight (HMW) DNAJC5 oligomers in the medium. Medium from HEK293T cell culture transfected with DNAJC5 was centrifuged at 1000 (1k)× g , 10,000 (10k)× g , and 100,000 (100k)× g , followed by SDS-PAGE and immunoblot of supernatant (s) fractions at each centrifugation step. ( B ) Fractionation of HMW-DNAJC5 with gel filtration. HEK293T cells transfected with DNAJC5 were lysed, clarified, and subjected to gel filtration. HMW-DNAJC5 of different sizes were separated based on their corresponding molecular weight. ( C ) XP-DNAJC5 L115R mutant forms a membrane-bound oligomer. Cellular fractionation was performed with HEK293T cells transfected with indicated DNAJC5 variants. Note the substantial change of electrophoretic mobility of XP-DNAJC5 L115R on SDS-PAGE. ( D ) α-syn secretion induced by XP-DNAJC5 L115R. Secretion assay was performed with HEK293T cells transfected with indicated plasmids. About 10 μm 2-BA was used to block induced α-syn. ( E ) Expression of α-syn and DNAJC5 variants in HEK293T cells. Note the substantial change in electrophoretic mobility of 2-BA-treated XP-DNAJC5 L115R on SDS-PAGE. Figure 6—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Molecular Weight, Cell Culture, Transfection, SDS Page, Western Blot, Centrifugation, Fractionation, Filtration, Mutagenesis, Membrane, Cell Fractionation, Blocking Assay, Expressing

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) DNAJC5 (ΔJ) forms a series of SDS-resistant oligomers. HEK293T cells transfected with WT DNAJC5 or DNAJC5 (ΔJ) were lysed and evaluated by immunoblot using anti-DNAJC5 antibody. ( B ) HMW-DNAJC5 is not formed by non-specific disulfide bonds. HEK293T cells transfected with DNAJC5 were lysed for SDS-PAGE followed by immunoblot. The loading samples for SDS-PAGE were prepared with increasing amount of DTT up to 40 mM. ( C ) Schematic diagram of XPACK. XPACK is myristoylated on the first glycine (G) and palmitoylated on the second cystine (C). Replacement of Serine (S) at position 5 with a hydrophobic isoleucine (I) abolishes normal lipidation of XPACK. ( D ) Assessment of different XPACK fusion constructs. Cell lysate containing different DNAJC5 constructs were evaluated by anti-DNAJC5 immunoblot. ( E ) Membrane association of XP-DNAJC5 dependent on palmitoylation. HEK293T cells transfected with XP-DNAJC5 were treated with DMSO or 10 μM 2-BA. After cell culture for 24 hr, cellular fractionation was performed with homogenized cells. Distribution of XP-DNAJC5 in cytosol (C) and membrane (M) fractions were evaluated with immunoblot. Figure 6—figure supplement 1—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Transfection, Western Blot, SDS Page, Construct, Membrane, Cell Culture, Cell Fractionation

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Internalization of punctate XP-DNAJC5 (green) into enlarged endosomes labeled by mCherry-Rab5 Q79L (red). U2OS cells were transfected with XP-DNAJC5 and mCherry-Rab5 Q79L . Live-cell imaging was performed 24 hr after transfection. Representative enlarged endosomes with XP-DNAJC5 are shown in magnified inset. Scale bar: 10 μm in overviews and 1 μm in magnified insets. ( B ) α-syn enters into enlarged endosomes in the presence of XP-DNAJC5. XP-DNAJC5-HaloTag (red) and mNG-α-syn (green) were coexpressed in U2OS cells carrying BFP-Rab5 Q79L (blue) mutant and imaged. Representative enlarged endosome with both XP-DNAJC5-HaloTag and mNG-α-syn inside is shown in magnified inset. Scale bar: 10 μm in overviews and 1 μm in magnified insets. ( C ) Quantification of the ratio of α-syn-containing endosomes in cells co-transfect with DNAJC5 L115R or cells co-transfected with DNAJC5 XP-L115R. More than 100 enlarged endosomes were counted in each group. Error bars represent standard deviations. P value<0.0001, two-tailed t test.

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Labeling, Transfection, Live Cell Imaging, Mutagenesis, Two Tailed Test

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Differentiation of SH-SY5Y was initiated by lowering the serum concentration to 1% FBS and addition of 10 µM retinoic acid (RA). Media were replaced every 3 days and the cells were harvested at indicated time to examine the expression of neuronal marker. DAT, dopamine transporter; Tuj1, neuron-specific class III β-tubulin. ( B ) In vitro depalmitoylation assay of endogenous DNAJC5 in differentiated SH-SY5Y cells. The depalmitoylation assay was performed as in using membrane (M) fraction from SH-SY5Y cells. HA, hydroxylamine. ( C ) Proteinase K protection assay of 100,000 (100k) pellet fraction from the centrifuged media of differentiated SH-SY5Y culture. Flot-2 and CD63 were used as exosome markers. ( D ) Sedimented α-syn was not buoyant. 100k pellet from ( C ) was mixed with 60% sucrose in PBS and layered with 40% and 20% sucrose in PBS sequentially. After centrifuged at 150,000× g for 16 hr, fractions were collected from top to bottom. Figure 7—figure supplement 1—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Concentration Assay, Expressing, Marker, In Vitro, Membrane, Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Membrane and cytosol fractionation of differentiated SH-SY5Y neuroblastoma cells. The fractionation was performed as depicted in . C, cytosol; M, membrane. The distribution of endogenous DNAJC5 and α-syn was evaluated by immunoblot. Transferrin receptor (TFR) was used as a membrane marker. Tubulin was used as a cytosol marker. ( B ) Quantification of α-syn level in the supernatant of centrifuged media with ELISA. Conditioned media were collected and sequentially centrifuged at 1000 (1k)× g , 10,000 (10k)× g , and 100,000 (100k)× g . The supernatant from each centrifugation step (1ks, 10ks, and 100ks) was collected and measured by LEGEND MAX Human α-synuclein (Colorimetric) ELISA Kit. One-way ANOVA showed no significant (ns) difference of α-syn level between fractions. ( C ) Quercetin inhibited endogenous α-syn secretion in hiPSC-derived midbrain dopamine neurons. hiPSC-dopamine neurons carrying the GBA-N370S mutation were treated with quercetin (5 μM or 10 μM) at day 35. Culture media samples were harvested after 3 days treatment at day 38 and α-syn levels in the media were analyzed by electro-chemiluminescent immunoassay. Data points represent individual cell lines derived from different donors and are normalised to total protein in the corresponding cell lysates. One-way ANOVA followed by Tukey’s post hoc test shows a significant reduction in α-syn secretion with increasing quercetin concentration (*p<0.05, **p<0.01). ( D ) Depletion of endogenous DNAJC5 in SH-SY5Y cells decreased basal α-syn secretion. After 3 days of culture, the media from differentiated SH-SY5Y cells transduced with shRNA targeting GFP (shRNA-GFP) or shRNA targeting DNAJC5 (shRNA-DNAJC5) were collected and the extracellular α-syn was quantified with ELISA. P value<0.0002, two-tailed t test. ( E ) Expression of exogenous human DNAJC5 in mouse mDA stimulated basal α-syn secretion. WT mDA and mDA expressing hDNAJC5 were treated with DMSO or 100 nM BaFA1. Quantification of α-syn in conditioned media was performed with Mouse α-synuclein ELISA Kit (Abcam). α-syn secretion was normalized by dividing the α-syn in media (pg/ml) by the α-syn in cell lysates (ng/ml). P value<0.01, one-way ANOVA. ( F ) BaFA1 increased DNAJC5 oligomerization in mouse mDA neurons. Figure 7—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Membrane, Fractionation, Western Blot, Marker, Enzyme-linked Immunosorbent Assay, Centrifugation, Derivative Assay, Mutagenesis, Concentration Assay, Transduction, shRNA, Two Tailed Test, Expressing

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Schematic of the differentiation protocol used to generate hiPSC-derived dopamine neurons, including a patterning phase to generate neural progenitor cells followed by differentiation into mature neurons. Green arrows represent points of replating when cells are supplemented with ROCK inhibitor (Ri) to increase survival. BDNF, brain-derived neurotrophic factor; FGF8a, fibroblast growth factor 8a; GDNF, glial cell line-derived neurotrophic factor; Puro, puromorphamine; SHH, sonic hedgehog; TGF-β3, transforming growth factor beta 3 (see ; ; for full details). Neurons are mature and harvested between 35 and 68 days for analysis. Immunocytochemical fluorescent images of mature neurons at day 50 stained for neuronal marker microtubule-associated protein (MAP2), the dopaminergic marker tyrosine hydroxylase (TH), and DAPI. Scale bar: 20 µm. ( B ) DNAJC5 is palmitoylated in iPSC-derived dopamine neurons. Immunoblot analysis of DNAJC5 palmitoylation in hiPSC-derived dopamine neurons treated with DMSO, 7.5 μM Quercetin or 10 μM 2-BA on day 65 and harvested on day 68. TH was used as a marker of dopaminergic identity. ( C ) Partial depalmitoylation of DNAJC5 by 2-BA in iPSC-derived dopamine neurons does not reduce α-syn secretion. hiPSC-derived dopamine neurons carrying the GBA-N370S mutation were treated with palmitoylation inhibitor 2-BA (5 μM or 10 μM) at day 35. Culture media samples were harvested after 3 days treatment at day 38 and α-syn levels in the media were analyzed by electro-chemiluminescent immunoassay. Data points represent individual cell lines derived from different donors and are normalized to total protein in the corresponding cell lysates. One-way ANOVA shows no effect of 2-BA on α-syn secretion. Figure 7—figure supplement 2—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Derivative Assay, Staining, Marker, Western Blot, Mutagenesis

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Examination of knockdown efficiency by shRNA targeting DNAJC5 in HEK293T cells. ( B ) Endogenous DNAJC5 expression decreased in SH-SY5Y cells transduced with shRNA targeting DNAJC5. Figure 7—figure supplement 3—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Knockdown, shRNA, Expressing, Transduction, Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Schematic overview of protocol used for differentiation of mESCs into middle-brain dopaminergic (mDA) neuronal cultures. Immunocytochemical staining using stem cell markers (Nanog, Sox2), neuronal precursor marker (Nestin), mDA markers [FoxA2, Nurr1, Lmx1a, Sox6 (selective marker of substantia nigra pars compacta lineage), TH (tyrosine hydroxylase), Girk2 (G-protein-regulated inward-rectifier potassium channel 2, expressed in DA neurons)], neuronal marker (Tuj1) and nuclear marker (dapi). ( B ) Expression of human DNAJC5 with C-terminal FLAG tag in mDA neurons. Figure 7—figure supplement 4—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Staining, Marker, Expressing, FLAG-tag, Western Blot

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Predicted structure of DNAJC5 by AlphaFold. Color scheme: J domain (magenta), Cys string domain (yellow) and C-terminal helix (green). ( B ) DNAJC5 (ΔJ) was competent to induce α-syn secretion into the medium. HEK293T cells were transfected with indicated plasmids. Media were collected after 36 hr and evaluated with immunoblot. ( C ) DNAJC5 (ΔJ) formed oligomers in HEK293T cells. ( D ) Quantification of normalized α-syn secretion in HEK293T cells transfected with WT DNAJC5 or DNAJC5 (ΔJ). Quantification was based on immunoblot in ( B ) and ( C ). The α-syn secretion was calculated as the amount of α-syn in media divided by the amount in lysate. α-syn secretion in cells transfected with WT DNAJC5 was normalized as 1. ( E ) C-terminal truncated DNAJC5 constructs were competent to induce α-syn secretion in the medium. HEK293T cells were transfected with C-terminal truncated DNAJC5 and α-syn. DNAJC5 antibodies cannot recognize DNAJC5 (ΔC30) and DNAJC5 (ΔC40) because of a missing epitope in the C-terminus. Instead, DNAJC5 (ΔC30) and DNAJC5 (ΔC40) were detected by C-terminal FLAG tags. All the C-terminal truncated DNAJC5 constructs showed smear-like oligomers. ( F ) Expression of C-terminal truncated DNAJC5 constructs in HEK293T cells. Immunoblot of anti-FLAG antibody and anti-DNAJC5 antibody cross-validated the existence of oligomers. ( G ) Quantification of normalized α-syn secretion in HEK293T cells transfected with WT DNAJC5 or different C-terminal truncated DNAJC5 constructs (ΔC10, ΔC20, ΔC30, and ΔC40). Quantification was based on immunoblot in ( E ) and ( F ). The α-syn secretion was calculated as the amount of α-syn in media divided by the amount in lysate. α-syn secretion in cells without DNAJC5 transfection was normalized as 1. Figure 8—source data 1. Uncropped immunoblot corresponding to .

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Transfection, Western Blot, Construct, Expressing

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet: ( A ) Recruitment of α-syn on the membrane by DNAJC5. DNAJC5 binds to α-syn and targets it to late endosomes by palmitoylation. DNAJC5 forms a high-order oligomer to accommodate α-syn. ( B ) Translocation of α-syn and DNAJC5 into the membrane compartment. Both α-syn and DNAJC5 are translocated into the endosome lumen along with intraluminal vesicles (ILVs), forming a multivesicular body (MVB). ( C ) Secretion of α-syn and DNAJC5. Upon fusion between MVB and plasma membrane (PM), the cargos are expelled into the extracellular space. α-syn is soluble. DNAJC5 exists in both soluble and membrane-bound forms. Further transmission potentially occurs after secretion.

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Membrane, Translocation Assay, Clinical Proteomics, Transmission Assay

Journal: eLife

Article Title: Unconventional secretion of α-synuclein mediated by palmitoylated DNAJC5 oligomers

doi: 10.7554/eLife.85837

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , CSP (DNAJC5) (NM_025219) Human Tagged ORF Clone , OriGene Technology , Cat# RC208826 , .

Techniques: Recombinant, Luciferase, Enzyme-linked Immunosorbent Assay, Cell Culture, Software