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