Journal: Human Molecular Genetics

Article Title: PD-linked CHCHD2 mutations impair CHCHD10 and MICOS complex leading to mitochondria dysfunction

doi: 10.1093/hmg/ddy413

Figure Lengend Snippet: PD-linked CHCHD2 mutants showed reduced binding to CHCHD10. ( A ) Co-immunoprecipitation of endogenous CHCHD2 by antibody against CHCHD2 in SK-N-SH cells. WCL: whole cell lysate, 5% total protein used in co-IP experiment. ( B ) Co-immunoprecipitation of endogenous CHCHD10 by antibody against CHCHD10 in SK-N-SH cells. WCL: whole cell lysate, 5% total protein used in co-IP experiment. ( C ) Co-immunoprecipitation of endogenous CHCHD2 by antibody against CHCHD2 in human brain tissue lysates. ( D ) Co-immunoprecipitation of CHCHD2 by a CHCHD2 antibody against middle region of CHCHD2 (labeled as CHCHD2-TF) in SK-N-SH cells transfected with non-tagged CHCHD2 WT/T61I/R145Q/Q126X. WCL: whole cell lysate, 5% total protein used in co-IP experiment. Lower arrow pointed to CHCHD2 Q126X and higher arrow pointed to full length CHCHD2. ( E ) Co-immunoprecipitation of CHCHD2 by a CHCHD2 antibody against middle region of CHCHD2 (labeled as CHCHD2-TF) in hESCs of H9 and isogenic lines harboring homozygous R145Q (−/−).WCL: whole cell lysate, 5% total protein used in co-IP experiment. Arrow pointed to CHCHD10. Representative results from R10 and R17 (R145Q−/−) were shown. ( F ) Quantification of protein abundance of CHCHD10 normalized with CHCHD2 on the co-immunoprecipitation complex from isogenic hESC lysates.

Article Snippet: Human brain tissue lysates were from Novus Centennial, CO. Elamipretide was from MedChemExpress Monmouth Junction, NJ.

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Labeling, Transfection, Quantitative Proteomics

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 3. A polyclonal N-terminal ADNP antibody from Aviva Systems detects ADNP specifically in murine and rat tissues and suggests proteolytic processing of the protein in the human brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats and humans were lysed in RIPA buffer and used as protein samples for the assessment of N-terminal antibody of Aviva systems. (A–C) The predicted molecular weight of ADNP is 124 kDa. The antibody recognizes ADNP in a range of 145 kDa with (E) additional lower mass signal of 85 kDa in all human brain regions. (B–D–F) Western blot analysis of the blocking peptide competition assay. Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced the signal observed at 145 kDa in all tested cell lines. Importantly, the 85 kDa band suggestive for proteolytic cleavage as well as degraded ADNP signal disappeared completely after immunization peptide supplementation. GAPDH was used as a loading control.

Article Snippet: Adult human brain (NB820-59177), human brain cerebellum (NB820-59180), human brain frontal lobe (NB82059186), and human brain hippocampus whole tissue lysates (NB820-59187) from a healthy 45-year-old male subject were purchased from Novus Biologicals.

Techniques: Control, Molecular Weight, Western Blot, Blocking Assay, Competitive Binding Assay, Concentration Assay

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 5. Different C-terminal ADNP antibodies detect ADNP in the range of 150 kDa and suggest proteolytic processing of the protein in the brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats, and humans were lysed in RIPA buffer and used as protein samples for the assessment with three C-terminal antibodies with the optimized dilutions listed in Table 3. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A)C) Murine samples indicate detection of ADNP in the range of 150 kDa with bands suggesting proteolytic processing at 50 kDa. (D–F) Rat samples indicate detection of ADNP in the range of 150 kDa with bands indicating proteolytic processing at 82 kDa after incubation with the C-terminal Abcam antibody. (G–I) Human brain samples indicate detection of ADNP at different molecular weights of 124 – 150 kDa in the adult frontal lobe and hippocampus and highlight the antibody differences in detection of ADNP. The three tested antibodies showed strong band signals at lower molecular weights, which could indicate proteolytic cleavage or degradation of the protein.

Article Snippet: Adult human brain (NB820-59177), human brain cerebellum (NB820-59180), human brain frontal lobe (NB82059186), and human brain hippocampus whole tissue lysates (NB820-59187) from a healthy 45-year-old male subject were purchased from Novus Biologicals.

Techniques: Control, Molecular Weight, Incubation

Journal: iScience

Article Title: Pulmonary surfactants and the respiratory-renal connection in steroid-sensitive nephrotic syndrome of childhood

doi: 10.1016/j.isci.2022.104694

Figure Lengend Snippet: Surfactant levels and urinary protein in SSNS/MCD (A) Plasma SP-A in control and ten subjects with SSNS/MCD in consecutive relapse/remission cycles (from 2 to 4 cycles). (B) Plasma SP-D in control and ten subjects with SSNS/MCD in consecutive relapse/remission cycles (from 2 to 4 cycles). (C) Correlation analysis between plasma SP-A and urinary SIRPα levels in samples from two controls (black symbols) and ten subjects with SSNS/MCD in relapse (blue symbols) and remission (red symbols). (D) Correlation analysis between plasma SP-A and urinary protein in samples from two controls (black symbols) and ten subjects with SSNS/MCD in relapse (blue symbols) and remission (red symbols). (E) Representative western blot for SP-A in the urine of a control subject (C1) and three patients with MCD in relapse (Rl1 to Rl3) and remission (Rm1 to Rm3). Statistical Analysis: One-Way ANOVA for A and B. For (C and D) Pearson r correlation analysis.

Article Snippet: Mouse SP-A (SFTPA) ELISA detection kit , Novus Biologicals , NBP2-77693.

Techniques: Clinical Proteomics, Control, Western Blot

Journal: iScience

Article Title: Pulmonary surfactants and the respiratory-renal connection in steroid-sensitive nephrotic syndrome of childhood

doi: 10.1016/j.isci.2022.104694

Figure Lengend Snippet: Intranasal instillation of LPS and PolyI:C and proteinuria in mice (A) Schematic depicting the protocol to induce proteinuria. Saline (red) or a combination of LPS (80 μg) and Poly I:C (240 μg) (blue) is administered intranasally once daily for three consecutive days. Urine is collected every 6 h and 3 h after the fourth injection, plasma, lung, and kidney tissue are collected for analysis. (B) Plasma SP-A levels in unmanipulated (black) mice or receiving either saline (red) or LPS/Poly I:C combination (n = 5 mice per group). (C) Representative confocal images for SP-A (green) and synaptopodin (red) colocalization in kidney glomeruli of mice receiving saline or LPS/Poly I:C. Merged colocalization is shown in yellow. Size bar: 200 μm. Images are representative from n = 5 mice per group (D) Confocal analysis of SIRPα (green) and SP-A (red) colocalization in kidney glomeruli of mice receiving saline or LPS/Poly I:C. Merged colocalization is shown in yellow. Size bar: 200 μm. Images are representative from five mice. (E) Co-immunoprecipitation of SIRPα and detection of SP-A in kidney cortex extracts of unmanipulated, saline-exposed, and LPS/Poly I:C-exposed mice. Total input (500 ug) and amount retained in preclearance is shown in lanes one and two; n = 3 mice per group. (F) Six hour albumin excretion over the 72-h period in unmanipulated (black) mice receiving either saline or LPS/Poly I:C. (G) 72-h accumulated albumin excretion in the urine of the same mice in F). n = 5 mice per group. (H) Representative electron microscopy podocyte images from saline- or LPS/PolyI:C-treated mice for 24 h. Blue arrows represent areas of foot effacement. Statistical Analysis: One-Way ANOVA, Tukey post hoc analysis for (B) and (G), data are represented as mean ± SEM.

Article Snippet: Mouse SP-A (SFTPA) ELISA detection kit , Novus Biologicals , NBP2-77693.

Techniques: Saline, Injection, Clinical Proteomics, Immunoprecipitation, Electron Microscopy

Journal: iScience

Article Title: Pulmonary surfactants and the respiratory-renal connection in steroid-sensitive nephrotic syndrome of childhood

doi: 10.1016/j.isci.2022.104694

Figure Lengend Snippet:

Article Snippet: Mouse SP-A (SFTPA) ELISA detection kit , Novus Biologicals , NBP2-77693.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay

Journal: Experimental Neurobiology

Article Title: LGR5 and Downstream Intracellular Signaling Proteins Play Critical Roles in the Cell Proliferation of Neuroblastoma, Meningioma and Pituitary Adenoma

doi: 10.5607/en.2019.28.5.628

Figure Lengend Snippet: Patient description and tumor sample characteristics

Article Snippet: Human brain whole tissue lysates and brain tissue slides from normal adults were obtained from Novus Biologicals (Littleton, CO, USA).

Techniques: Concentration Assay

Journal: Experimental Neurobiology

Article Title: LGR5 and Downstream Intracellular Signaling Proteins Play Critical Roles in the Cell Proliferation of Neuroblastoma, Meningioma and Pituitary Adenoma

doi: 10.5607/en.2019.28.5.628

Figure Lengend Snippet: Expression of LGR5, hnRNPA2B1, and hnRNPH3 proteins in human normal, benign meningioma, and pituitary adenoma tissues, and immunohistochemistry for LGR5 and hnRNPH3 in meningioma and pituitary adenoma tissues with immunofluorescence confocal microscopy. (A) Comparison of expression levels of LGR5, hnRNPA2B1, and hnRNPH3 between normal, benign meningioma, and pituitary adenoma by western blotting. (B) Confocal images of the human normal brain (top), meningioma (middle) and pituitary adenoma (bottom) tissues stained with antibodies against LGR5 (green) and hnRNPH3 (red). Scale bar: 20 μm. The number of LGR5 + /hnRNPH3 + cells per DAPI positive cells and hnRNPH3 + cells per LGR5 positive cells was counted and expressed as %.

Article Snippet: Human brain whole tissue lysates and brain tissue slides from normal adults were obtained from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Confocal Microscopy, Comparison, Western Blot, Staining