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: Oxidative Medicine and Cellular Longevity

Article Title: Pterostilbene Confers Protection against Diquat-Induced Intestinal Damage with Potential Regulation of Redox Status and Ferroptosis in Broiler Chickens

doi: 10.1155/2023/8258354

Figure Lengend Snippet: Effects of pterostilbene on mitochondrial redox status and function in the jejunum of diquat-challenged broilers. (a) Mitochondrial ROS in the jejunum was measured by a fluorescence probe DHE. (b) Jejunal SOD2 activity. (c–f) The jejunal activities of mitochondrial complexes I, III, and IV and ATP synthase. (g) Jejunal ATP level. (h) Jejunal mtDNA content. (i) qRT-PCR analysis was conducted to detect the expression of genes related to mitochondrial biogenesis in the jejunum. Data are shown as mean ± standard error, n = 6/group; ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: After blocking with a QuickBlockTM Blocking Buffer for Western blot (#P0252; Beyotime), the PVDF membranes were washed with the Tris-buffered saline containing Tween-20 (TBST) and incubated with the primary antibodies against OCLN (1 : 3,000; #13409-1-AP; Proteintech; Rosemont, IL, USA), ZO-1 (1 : 1,000; #21773-1-AP; Proteintech), GPX4 (1 : 3,000; #67763-1-Ig; Proteintech), SLC7A11 (1 : 1,000; #A13685; ABclonal, Wuhan, Hubei, China), FTH1 (1 : 1,000; #A19544; ABclonal), ACSL4 (1 : 1,000; #A6826; ABclonal), NRF2 (1 : 1,000; #16396-1-AP; Proteintech), HO1(1 : 1,000; #27282-1-AP; Proteintech), SOD2 (1 : 5,000; #24147-1-AP; Proteintech), lamin B1 (1 : 5,000; #12987-1-AP; Proteintech), and β -actin (1 : 10,000; #60004-1-Ig; Proteintech) at 4°C overnight.

Techniques: Fluorescence, Activity Assay, Quantitative RT-PCR, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pterostilbene Confers Protection against Diquat-Induced Intestinal Damage with Potential Regulation of Redox Status and Ferroptosis in Broiler Chickens

doi: 10.1155/2023/8258354

Figure Lengend Snippet: Pterostilbene activates NRF2 signals in the jejunum of diquat-challenged broilers. (a–d) Western blot analysis was conducted to determine the protein levels of nuclear NRF2, HO1, and SOD2 in the jejunum. (e) qRT-PCR analysis was conducted to detect the mRNA expression of NRF2 targets in the jejunum. Data are shown as mean ± standard error, n = 6/group; ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: After blocking with a QuickBlockTM Blocking Buffer for Western blot (#P0252; Beyotime), the PVDF membranes were washed with the Tris-buffered saline containing Tween-20 (TBST) and incubated with the primary antibodies against OCLN (1 : 3,000; #13409-1-AP; Proteintech; Rosemont, IL, USA), ZO-1 (1 : 1,000; #21773-1-AP; Proteintech), GPX4 (1 : 3,000; #67763-1-Ig; Proteintech), SLC7A11 (1 : 1,000; #A13685; ABclonal, Wuhan, Hubei, China), FTH1 (1 : 1,000; #A19544; ABclonal), ACSL4 (1 : 1,000; #A6826; ABclonal), NRF2 (1 : 1,000; #16396-1-AP; Proteintech), HO1(1 : 1,000; #27282-1-AP; Proteintech), SOD2 (1 : 5,000; #24147-1-AP; Proteintech), lamin B1 (1 : 5,000; #12987-1-AP; Proteintech), and β -actin (1 : 10,000; #60004-1-Ig; Proteintech) at 4°C overnight.

Techniques: Western Blot, Quantitative RT-PCR, Expressing