Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Subcellular localization of Luman during osteoclastogenesis. BMMs were infected with a retroviral vector expressing FLAG-tagged Luman. After viral infection, BMMs were cultured with M-CSF for 2 days. Thereafter, infected BMMs were incubated with M-CSF and RANKL for the indicated time periods. Cells were treated with proteasome inhibitor MG132 4 h before fixation, and then were fixed with cold methanol. Immunostaining was performed with antibodies against FLAG and calnexin (CNX), an ER marker. Nuclear counter-staining was conducted with DAPI. At day 2, signals for Luman in the nucleus as well as in the ER were detected (arrows). Scale bar: 20 µm.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Infection, Retroviral, Plasmid Preparation, Expressing, Cell Culture, Incubation, Immunostaining, Marker, Staining

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Luman mediates the induction of DC-STAMP through CRE-like sequence in the DC-STAMP promoter. (A) Schematic representation of the 0.2-kb promoter region of the murine DC-STAMP gene. AP-1 site (△), NFAT site (□) and CRE-like sequence site (◆) are indicated. TSS, transcription start site. Each CRE-like sequence is indicated. (B) The reporter plasmid of the 0.2-kb murine DC-STAMP promoter fused with the luciferase gene was co-transfected with the pcDNA empty vector (Mock) or Luman N-terminus pcDNA expression vector (Luman-N) into RAW264 cells. Luciferase activity was measured at 24 h after transfection. Data are from three independent experiments. Values indicate mean±s.e.m. † P <0.001. (C) Luciferase reporter assay with a series of deletion mutants of the DC-STAMP promoter reporter plasmids. Reporter plasmids in which the NFAT-binding site or each CRE-like sequence had been deleted from the DC-STAMP promoter region were used. Reporter activities were measured in the same way as described in B. Data are from three independent experiments. Values indicate mean±s.e.m. ** P <0.01; † P <0.001; n.s., not significant. (D) Electrophoretic mobility shift assay. The biotin-labeled probes, including the CRE(2) site of the DC-STAMP promoter, were incubated with nuclear extract derived from FLAG–Luman-N expressing HeLa cells. Note that the binding of FLAG–Luman-N to the CRE(2) site was abolished by a competitor (lane 3), and a supershift after incubation with anti-FLAG antibodies (lane 4) was detected.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Sequencing, Plasmid Preparation, Luciferase, Transfection, Expressing, Activity Assay, Reporter Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Derivative Assay

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Luman interacts with DC-STAMP and is localized to the Golgi. (A) Co-immunoprecipitation analyses of HeLa cells expressing Luman and DC-STAMP. Cells were co-transfected with expression plasmids for Luman tagged with FLAG at the N-terminus (FLAG–Luman) and DC-STAMP tagged with HA at the C-terminus (DC-STAMP–HA). Cell lysates were immunoprecipitated with antibodies against HA (a-HA), and the immunoprecipitated (IP) samples were subjected to western blotting (WB) with an antibody against FLAG. (B) Subcellular localization of Luman and DC-STAMP that had been expressed in HeLa cells. FLAG–Luman and/or DC-STAMP–HA expression plasmids were transfected into HeLa cells. The transfected cells were immunostained with antibodies against FLAG or HA. Note that co-expression of Luman and DC-STAMP caused the emergence of perinuclear accumulation. Arrows indicate accumulated signals of Luman and DC-STAMP. Scale bars: 10 µm. (C) Double-staining for FLAG and Golgi markers on the cells expressing FLAG–Luman and DC-STAMP–HA. HeLa cells were co-transfected with FLAG–Luman and DC-STAMP–HA expression plasmids. The transfected cells were immunostained with antibodies against FLAG and GM130 (cis-Golgi marker) or against FLAG and TGN46 (trans-Golgi marker). Note that perinuclear accumulation of FLAG–Luman and DC-STAMP–HA overlaps with that of the trans-Golgi marker. Arrows indicate accumulated signals for Luman and DC-STAMP. Scale bars: 10 µm.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Immunoprecipitation, Expressing, Transfection, Western Blot, Double Staining, Marker

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Luman defines the localization and stabilization of DC-STAMP by interacting with it. (A) Schematic representation of the domain structure of murine DC-STAMP and mapping of the binding region for Luman. Co-immunoprecipitation experiments with various truncated DC-STAMP mutants revealed the binding region in DC-STAMP for Luman. Solid squares with Roman numerals indicate the transmembrane domains of DC-STAMP. Putative binding regions are highlighted in red. The degrees of association between Luman and the DC-STAMP constructs shown are indicated as follows: circle, strong; triangle, weak; cross, none. Numbers represent amino acid residues. (B) Co-immunoprecipitation followed by western blot analysis. HeLa cells were co-transfected with expression plasmids for FLAG–Luman and full-length DC-STAMP [DC-STAMP(full)–HA] or HA-tagged truncated DC-STAMP [DC-STAMP(1-167)–HA]. Cell lysates were immunoprecipitated (IP) with antibodies against HA (a-HA), and the immunoprecipitated proteins were subjected to western blotting with antibodies against FLAG or HA. Note that Luman only interacts with the full-length DC-STAMP. (C) Immunostaining for FLAG–Luman and DC-STAMP–HA. HeLa cells were co-transfected with FLAG–Luman and DC-STAMP(full)–HA or DC-STAMP(1-167)–HA expression plasmids, and immunostained with antibodies against FLAG (green) and HA (red). Upon co-expression of FLAG–Luman and DC-STAMP(full)–HA, both proteins accumulated at the perinuclear region. DC-STAMP(1-167)–HA was scarcely detected in the absence of treatment with MG132. Arrows indicate accumulated signals for Luman and DC-STAMP. Scale bars: 10 µm. (D) Subcellular fractionation analyses of Luman and DC-STAMP. HeLa cells were co-transfected with expression plasmids for FLAG–Luman and DC-STAMP(full)–HA or DC-STAMP(1-167)–HA. The microsome membranes from cell lysates were ultracentrifuged and fractionated in an iodixanol gradient. Each fraction was subjected to western blotting. TGN46, GM130 and calnexin (CNX) were examined as specific intracellular markers for the trans-Golgi, cis-Golgi and ER, respectively. TGN46 is mainly distributed in fractions 2 and 3; GM130 in fractions 4–6; CNX in fractions 7–9.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Binding Assay, Immunoprecipitation, Construct, Western Blot, Transfection, Expressing, Immunostaining, Fractionation

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 6. Unambiguous detection of ADNP using homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines. mESCs containing either wild-type, homozygous mutants, or complete Adnp knockout were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal ADNP, 3x-DYKDDDDK, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight with additional lower mass signal of 37—65 kDa in Adnp homozygous and parental control mESCs. (B) Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced all signals observed mESC lines, indicating that the N-terminal antibody does not bind ADNP specifically in mESCs. (C) Detection of wild- type and homozygous Adnp mutants by means of a C-terminal 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 150 kDa in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line using a DYKDDDDK antibody. Truncated ADNP mutants, p.Tyr718* and p.Lys407Valfs*31, were detected at a lower molecular weight of 80 kDa, respectively 48 kDa. (D–F) Wild-type ADNP detection by means of three different C-terminal antibodies in mESC lines. Wild-type ADNP was detected with a strong signal at 150 kDa in the parental control line with a rather decreased signal in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line. Disappearance of the 150 kDa band was observed in the mESC line with complete Adnp homozygosity, indicating a reliable molecular weight of 150 kDa for ADNP.

Article Snippet: Protein lysates of HEK293T cells transfected with either wild-type or mutated ADNP constructs were analyzed by immunoblotting for anti-GFP, anti-DYKDDDDK (Flag), and the N-terminal antibody of Aviva Systems, since Helsmoortel-Van der Aa mutations are characterized by a premature stop codon truncating the C-terminus of ADNP14.

Techniques: CRISPR, Knock-Out, Control, Molecular Weight, Concentration Assay, FLAG-tag

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 7. Unambiguous detection of ADNP using an N-terminal GFPSpark and N-DYKDDDDK (Flag) tag expression vector. (A) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP- GFPSpark and mutated constructs using an anti-GFP antibody. (B) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-GFPSpark and mutated constructs using the N-terminal ADNP antibody (Aviva Systems). (C) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP- DYKDDDDK (Flag) and mutated constructs using an anti-DYKDDDDK antibody. (D) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-DYKDDDDK and mutant constructs using the N-terminal ADNP antibody (Aviva Systems). The observed molecular weight of wild-type ADNP-GFPSpark is 175 kDa (including 25 kDa GFPSpark tag), respectively ADNP-DYKDDDDK 150 kDa, with each of their mutants showing a lower molecular weight as a consequence of the truncating mutations. Detection with antibodies for GFP, DYKDDDDK (Flag), and ADNP gave comparable results. GAPDH was used as a loading control in all experiments.

Article Snippet: Protein lysates of HEK293T cells transfected with either wild-type or mutated ADNP constructs were analyzed by immunoblotting for anti-GFP, anti-DYKDDDDK (Flag), and the N-terminal antibody of Aviva Systems, since Helsmoortel-Van der Aa mutations are characterized by a premature stop codon truncating the C-terminus of ADNP14.

Techniques: FLAG-tag, Expressing, Plasmid Preparation, Western Blot, Construct, Mutagenesis, Molecular Weight, Control

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 8. Western blotting of ADNP in a HCT116 colon cancer cell line, carrying the prevalent heterozygous p.Tyr719* mutation. HCT116 cells containing a wild-type and p.Tyr719* mutant allele were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal antibody, 3x-DYKDDDDK, HA-tag, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control in all experiment. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight an additional signal of 45 kDa, indicating proteolytic cleavage or non-specific binding. (B) Administration of the immunization peptide in a 5 × excess to antibody concentration reduced all signals, indicating that the N-terminal antibody does not bind ADNP specifically in HCT116 cells. (C) Detection of wild-type ADNP by means of the 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 182 kDa in the 3xFlag-V5-loxP- neonGreen/3xHA-loxP-mCherry engineered line using a DYKDDDDK antibody, 32 kDa by tag insertion. (D) Detection of mutant ADNP by means of the HA-epitope tag. A truncated mutant p.Tyr719 ADNP protein was detected in at 105 kDa in the 3xFlag-V5-loxP-neonGreen/3xHA-loxP-mCherry engineered line using a HA-antibody, 25 kDa above its predicted molecular weight by tag insertion. Instability of the truncated protein was observed by a degrading smear. (E–G) Wild-type ADNP detection by means of three different C-terminal antibodies. Non-processed ADNP was detected with a strong signal at 150 kDa in the control line and at a molecular weight of 182 kDa in the genome-edited cell line. In both cases, a degrading smear was observed, indicating instability of the wild-type protein.

Article Snippet: Protein lysates of HEK293T cells transfected with either wild-type or mutated ADNP constructs were analyzed by immunoblotting for anti-GFP, anti-DYKDDDDK (Flag), and the N-terminal antibody of Aviva Systems, since Helsmoortel-Van der Aa mutations are characterized by a premature stop codon truncating the C-terminus of ADNP14.

Techniques: Western Blot, Mutagenesis, Control, Molecular Weight, Binding Assay, Concentration Assay, FLAG-tag

Journal: Nature chemical biology

Article Title: A chemoproteomic portrait of the oncometabolite fumarate

doi: 10.1038/s41589-018-0217-y

Figure Lengend Snippet: Functional analyses of FH -regulated Cys residues. (a) Percentage of FH -regulated Cys residues predicted to be functional using the informatics tool Mutation Assessor (R ≥1.5, n ≥2). (b) Gene ontology analysis of FH -regulated Cys residues. (c) Correlation between genes containing FH -regulated Cys residues and genetic alterations in kidney cancer (RCC). Statistical significance was assessed using Student’s t -test (two-tailed, unpaired); ** P < 0.01. (d) SMARCC1 C520 lies in the SWIRM domain and is highly conserved. (e) SMARCC1 C520 lies at the SNF5 subunit interface. (f) SMARCC1 C520 undergoes FH -regulated changes in occupancy in HLRCC cells ( n = 3 independent experiments). (g) SMARCC1 C520E mutation limits co-immunoprecipitation with SNF5 in HEK-293 cells co-overexpressing FLAG-tagged SNF5 with Myc-tagged SMARCC1. (h) SMARCC1 S-succination can be detected in FH-deficient and FH WT HLRCC cell lines after IP of endogenous SMARCC1. (i) SNF5 demonstrates decreased co-immunoprecipitation and decreased levels in FH−/− HLRCC cells. Left: Results from SWI/SNF complex co-immunoprecipitation with BRG1 antibody. Right: Endogenous levels of SWI/SNF complex members in HLRCC cells. Representative images from two independent experiments are shown in g-i. Uncropped scans of immunoblots are provided in . (j) EZH2 inhibitors exhibit modest selectivity for FH-deficient HLRCC cells. UOK262 FH −/− or FH WT spheroids were treated with GSK126 (21 days; 1, 2, 4, 8, 16 μM) and % viability plotted relative to the vehicle treated spheroids. Data is presented as mean ± s.d.; n = 3/group. (k) EZH2 inhibitors are toxic to HLRCC spheroids. UOK262 FH −/− spheroids were treated with vehicle or EPZ6438 (14 days; 5 μM). Figure is representative of 6 replicates.

Article Snippet: Anti-FLAG pulldown was performed using immunoprecipitation kit (KBA-319–383) from Rockland Immunochemicals, Inc. SDS-PAGE was performed using Bis-Tris NuPAGE gels (4–12%, Invitrogen #NP0322), and MES running buffer (Life technologies #NP0002) in Xcell SureLock MiniCells (Invitrogen) according to the manufacturer’s instructions.

Techniques: Functional Assay, Mutagenesis, Two Tailed Test, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: A developmental framework linking neurogenesis and circuit formation in the Drosophila CNS

doi: 10.7554/eLife.67510

Figure Lengend Snippet:

Article Snippet: Antibody, polyclonal , Rabbit anti-FLAG Dylite488 # 600-441-383 , Rockland, Atlanta, GA , RRID: AB_1961508 , 1:200.

Techniques: Marker, Clone Assay, CRISPR, Labeling, Sequencing