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: Science Advances

Article Title: Norovirus co-opts NINJ1 for selective protein secretion

doi: 10.1126/sciadv.adu7985

Figure Lengend Snippet: ( A ) WT or Ninj1 KO BV2 cells were infected with CR6 at 5 MOI and harvested at 10 hpi. Cell lysates were immunoprecipitated with anti-NS1 and analyzed by immunoblotting. ( B and C ) Transmission electron microscopy image of uninfected (B) or CR6-infected (C) BV2 cells with immunogold labeling for FLAG-NINJ1 (12-nm gold, black arrowheads) and NS1 (18-nm gold, red arrowheads). RC, replication complex. Scale bar, 500 nm. ( D ) Representative immunofluorescence confocal imaging of FLAG-NINJ1 (red), NS1 (green), NS6/7 (cyan), and 4′,6-diamidino-2-phenylindole (DAPI, blue) in Ninj1 KO BV2 cells reconstituted with FLAG-NINJ1 WT or K45Q at 14 hpi. White arrowhead highlights the colocalization of NINJ1 and NS1 proteins. Scale bars, 5 μm. All images are representative of three or more independent experiments. ( E ) Quantification of colocalization between NS1 and FLAG-NINJ1 WT or K45Q. Bars represent means ± SEM, n = 10 to 11 cells. ( F ) Dose-dependent binding of Fc-tagged ectodomain of human NINJ1 (hNINJ1) or mouse NINJ1 (mNINJ1) protein to recombinant NS1 protein was determined by ELISA. A monoclonal anti-NS1 (clone CM79) and isotype control were included as a positive and negative control, respectively. Data represent means ± SEM of duplicates, and the dissociation constants ( K d ) are shown. ( G to I ) Direct binding between recombinant NS1 and mNINJ1 protein was measured by MST. ΔFnorm indicates the change in thermophoresis, and K d is shown. n.d., not determined. (G) Dose-response curve showing bindings of WT NS1 protein to fluorescence-labeled WT or mutated mNINJ1 proteins. (H) Dose-response curve showing bindings of WT or mutated NS1 proteins to fluorescence-labeled mNINJ1. Maltose-binding protein (MBP) was included as a negative control. (I) Dose-response curve showing bindings of WT or mutated NS1 proteins to fluorescence-labeled monoclonal anti-NS1 or isotype control. Data are shown in means ± SEM ( n = 3).

Article Snippet: Primary antibodies used in this study include the following: Strep-tag II (A01732, Genscript), FLAG (for immunoblotting; 200-350-383, Rockland Immunochemicals), FLAG (for immunofluorescence; 637303, BioLegend), 6xHis (ab18184, Abcam), GSDMD (ab209845, Abcam), active caspase-3 (clone C92-605; 559565, BD Biosciences), glyceraldehyde-3-phosphate dehydrogenase (MCA4739, Bio-Rad), calnexin (ab22595, Abcam), RAB7 (ab137029, Abcam), ATP5A1 (14676-1-AP, Proteintech), and TGN46 (ab16059, Abcam).

Techniques: Infection, Immunoprecipitation, Western Blot, Transmission Assay, Electron Microscopy, Labeling, Immunofluorescence, Imaging, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Negative Control, Fluorescence

Journal: Advanced Science

Article Title: G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC‐I via PI3K/Akt Activation in Bladder Cancer

doi: 10.1002/advs.202305922

Figure Lengend Snippet: G3BP1 interacts with SLU7 to promote PI3K/Akt signaling activation, downregulating MHC‐I and promoting immune evasion. A,B) qRT–PCR analysis was used to evaluate the mRNA levels of SLU7 and the class IA PI3Ks in UMUC3 and T24 cells with SLU7 knockdown or overexpression. C,D) Western blotting was used to detect the protein expression levels of class IA PI3Ks in UMUC3 and T24 cells with SLU7 knockdown or overexpression. E) Cell lysates from UMUC3 cells with stable expression of SLU7‐Flag or HA‐G3BP1 were immunoprecipitated with anti‐Flag or anti‐HA antibodies, and immunoblotting was conducted with the indicated antibodies. F) The PLA was used to evaluate the interaction between G3BP1 and SLU7 in UMUC3 cells. Scale bar, 10 µm. G) IF was used to analyze the locations of G3BP1 and SLU7 in UMUC3 cells. Scale bar, 10 µm. Fluorescence intensity curves of DAPI (blue), G3BP1 (green), and SLU7 (red) that crossed the arrow were analyzed. H,I) The protein expression levels of SLU7, total Akt, p‐Akt Thr308 , and p‐Akt Ser473 in UMUC3 and MB49 cells with stable SLU7 knockdown were detected using western blotting. J,K) Western blotting was used to detect the protein expression levels of G3BP1, SLU7, total Akt, p‐Akt Thr308 , p‐Akt Ser473 , and/or MHC‐I in G3BP1‐overexpressing UMUC3 and MB49 cells with SLU7 knockdown. L) Flow cytometry was used to analyze the expression of MHC‐I on the surface of MB49 cells in (K). MHC‐I expression was quantified based on the mean fluorescence intensity (MFI). M) mCherry‐expressing MB49‐OVA cells with or without G3bp1 overexpression and Slu7 knockdown were cocultured with antigen‐activated CTLs for 24 h. Cytotoxicity was determined by analyzing the percentage of 7‐AAD+ cells among mCherry+ cells. Error bars represent SD ( n = 3 in A, B, L, and M). P ‐values are presented and were calculated using two‐way ANOVA (A and B) and one‐way ANOVA (L and M).

Article Snippet: For re‐IP, UMUC3 cell lysates with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody, after which they were blocked with 15 μg mL −1 Flag peptide (TP1273, TargetMol, USA) for 2 h at 4°C under gentle rotation.

Techniques: Activation Assay, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Expressing, Immunoprecipitation, Fluorescence, Flow Cytometry

Journal: Advanced Science

Article Title: G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC‐I via PI3K/Akt Activation in Bladder Cancer

doi: 10.1002/advs.202305922

Figure Lengend Snippet: G3BP1 and SLU7 form a complex with PABPC1 and eIF4G1 to stabilize the closed‐loop structure of the mRNAs of class IA PI3Ks. A) Scatter plot of the G3BP1‐interacting proteins identified using MS. B and C) Cell lysates from UMUC3 cells with stable expression of SLU7‐Flag or HA‐G3BP1 were immunoprecipitated with anti‐Flag or anti‐HA antibodies, and immunoblotting was performed with the indicated antibodies. D) Cell lysates from UMUC3 cells with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody and then blocked with Flag peptide. The resultant immunoprecipitants were subjected to re‐IP with anti‐HA antibodies and immunoblotted with the indicated antibodies. E) IF was used to analyze the locations of eIF4G1 or PABPC1 with SLU7 and G3BP1 in UMUC3 cells stably expressing eIF4G1‐GFP or PABPC1‐GFP. Scale bar, 10 µm. The fluorescence intensity curves of DAPI (blue), eIF4G1‐GFP (green), PABPC1‐GFP (green), SLU7 (red), and G3BP1 (purple) that crossed the arrows were analyzed. F) Cell lysates from UMUC3 cells with PABPC1‐GFP stable expression and SLU7 or G3BP1 knockdown were immunoprecipitated with anti‐GFP antibody. Western blotting was used to detect the expression levels of PABPC1‐GFP, SLU7, G3BP1, and eIF4G1 in whole cell lysates and immunoprecipitants. G) qRT–PCR analysis was used to evaluate the mRNA expression levels of class IA PI3Ks in the immunoprecipitants. H) Western blotting was used to detect the expression levels of G3BP1 and SLU7 in UMUC3 cells with SLU7 knockdown or G3BP1 knockdown. I) FISH–FRET was performed in UMUC3 cells with SLU7 or G3BP1 knockdown using Cy3‐labeled 5′‐UTR probes and Cy5‐labeled 3′‐UTR probes targeting the mRNAs of PI3Ks. The mean values of FRET efficiency are shown. Error bars represent SD ( n = 3 in G; n = 5 in I). P ‐values are presented and were calculated using one‐way ANOVA (G and I).

Article Snippet: For re‐IP, UMUC3 cell lysates with simultaneous stable expression of SLU7‐Flag and HA‐G3BP1 were primarily immunoprecipitated with anti‐Flag antibody, after which they were blocked with 15 μg mL −1 Flag peptide (TP1273, TargetMol, USA) for 2 h at 4°C under gentle rotation.

Techniques: Expressing, Immunoprecipitation, Western Blot, Stable Transfection, Fluorescence, Knockdown, Quantitative RT-PCR, Labeling

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Role of the di-proline motif in degradation of Δ9-desaturase. N-terminal amino acid sequences of Δ9-desaturases were compared (D. melanogaster DESAT1 (NP_652731), Mus musculus SCD1 (NP_033153), M. musculus SCD3 (NP_077770), Homo sapiens SCD1 (NP_005054), Danio rerio stearoyl-CoA desaturase (AAO25582), and S. cerevisiae Ole1 (CAA96757)) (A). Schematic illustration of DESAT1, mouse SCD1, and constructed mutants is shown (B). S2 cells expressing mouse SCD1-FLAGC, chimera-FLAGC, chimera (AA)-FLAGC, and SCD1 (di-Pro)-FLAGC were treated with C18:1 (100 μm) for 6 h (C) or DESAT1 inhibitor 37c (1 μm) for 16 h (D), and the amounts of endogenous DESAT1, FLAG-tagged exogenous Δ9-desaturases, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively. Band intensities were determined by ImageJ software, and levels of SCD1 proteins are shown relative to the amount of SCD1 protein in vehicle-treated cells (C and D). Mean ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Construct, Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Identification of protease involved in the degradation of DESAT1. S2 cells were treated with calpeptin (50 μm), MG132 (50 μg/ml), or chloroquine (50 μm) for 6 h (A). S2 cells were treated with dsRNA against GFP, CalpA, CalpB, CalpC, or sol for 72 h and then exposed to C18:1 (100 μm) for 6 h (B). S2 cells expressing DESAT1-FLAGC and DESAT1 (P2A/P3A)-FLAGC were treated with calpeptin (50 μm) for 6 h (C). The amounts of endogenous DESAT1, FLAG-tagged exogenously expressed DESAT1, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively (A–C). Band intensities were determined by ImageJ software, and levels of DESAT1 proteins are shown relative to the amount of DESAT1 protein in vehicle-treated cells (A–C). Mean ± S.D. (n = 3). **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Expressing, Software