Journal: bioRxiv

Article Title: Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

doi: 10.1101/232397

Figure Lengend Snippet: (A) Schematic of RPE differentiation timeline; (B, C) Bright field images and immunostaining for RPE markers; (D) Correct basolateral distribution of collagen IV (CIV) and MERTK in unaffected control (WT3) but not RP11 RPE cells; (E, F) ELISA assays for basal and apical secretion of VEGF and PEDF respectively in control and RP11 RPE cells. (G, H) Fluid transport and trans-epithelial resistance measurement revealed a significant difference between patient and RP11 RPE cells; (I) Reduced phagocytic capacity in RP11 RPE cells. E-I: Data shown as mean ± SEM, n=3. Statistical significance of pair-wise comparisons is indicated by n.s. not significant; *** p<0.001; **** p<0.0001 (Student’s paired t-test).

Article Snippet: Cells were treated with primary antibodies Anti-Bestrophin (Abcam, 1:300), Anti-Sodium Potassium ATPase (Alexa Fluor ® 488 conjugate) (Abcam, 1:50), Pericentrin (Abcam, 1:500), MERTK (Bethyl, 1:200), ARL13B (Proteintech, 1:500), Collagen IV (Abcam, 1:200), PRPF31 (Abnova, 1:500) and SNRPB Monoclonal Antibody (Y12) (Thermo, 1|:500), overnight at 4°C, and with secondary antibodies anti-rabbit FITC (Sigma, 1:500) or anti mouse FITC (Jackson Immuno Research, 1:500) and anti-mouse Cy3 (Jackson Immuno Research, 1:500) or anti-rabbit Cy3 (Jackson Immuno Researchl:500) diluted in PBS for 1 hour at room temperature.

Techniques: Immunostaining, Control, Enzyme-linked Immunosorbent Assay

Journal: Translational Neurodegeneration

Article Title: Attenuating α-synuclein pathology in mice with in situ engineered astrocytes

doi: 10.1186/s40035-025-00518-0

Figure Lengend Snippet: Expression of CAR-A and effect on α-synO phagocytosis and digestion. a Design of the CAR-expression plasmid. CAR was expressed in fusion with 3A scFv and enhanced green fluorescent protein (EGFP) under the control of the astrocyte-specific promotor GfaABC1D. SP, signal peptide; Poly(A), polyadenylation signal; ORI, origin of replication; KanR, kanamycin resistance gene. b Representative image of CAR expression on an astrocyte. The co-localization of 3A, MerTK and EGFP was assessed by confocal microscopy. Scale bars, 10 μm. c Flow cytometry analysis of the binding of CAR-A and ns-CAR-A to α-syn monomers and oligomers (α-synOs). The astrocytes were transfected with CAR or ns-CAR lipoplexes for 48 h. After 2 h-incubation with 1 μmol/L α-syn monomers (α-syn) or α-synOs, cells were stained with PE-labeled anti-α-syn antibody. d PE fluorescence in EGFP-positive astrocytes. n = 3 independent experiments. e Flow cytometry analysis of the amount of α-synO engulfed by CAR-A, ns-CAR-A and NC-A in the presence of different α-synO concentrations. n = 3 independent experiments. f Representative images depicting the phases of engulfment and digestion of α-synO by CAR-A. CAR-A was treated with 1 μmol/L α-synO, and the medium was changed after 1 h incubation. α-SynO and Lamp1 in CAR-A were stained with respective antibodies at different time points and imaged by confocal microscopy. Scale bars, 5 μm. g The kinetic curves of α-synO digestion in CAR-A, ns-CAR-A and NC-A. n = 3 independent experiments. h Statistical analysis of the proportion of α-syn colocalized with Lamp1 in digestion stage in ( f ) by Image J. n = 4 independent experiments. i Intracellular α-syn in Triton X-100-soluble and -insoluble fraction detected by Western blotting at different time points post astrocytic phagocytose of α-synOs. β-actin was used as a control. j Quantification of α-syn ( i ) using Image J. n = 3 independent experiments. k Representative images depicting the binding of ns-CAR-A, NC-A and CAR-A to α-syn monomers and oligomers. Scale bars, 5 μm. Data are mean ± S.E.M. One-way ANOVA ( d ) or Two-way ANOVA ( e ) followed by Tukey’s multiple comparison test was used for statistical analysis. * P < 0.05, ** P < 0.01, **** P < 0.0001 indicate significance compared to respective groups

Article Snippet: In the first construct, the enhanced CMV promotor sequence in the MerTK expression plasmid (purchased from Sino Biological Inc., #MG50514-ACG) was replaced with GfaABC1D promotor sequence synthesized from Sangon.

Techniques: Expressing, Plasmid Preparation, Control, Confocal Microscopy, Flow Cytometry, Binding Assay, Transfection, Incubation, Staining, Labeling, Fluorescence, Western Blot, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: Photo-affinity conjugation of Herceptin with homophilic peptide at different ratios. Tryptophan containing homophilic peptide was added to Herceptin at different amounts and photo-crosslinked as described [17]. The conjugate was applied to Sephacryl S300 in PBS containing 2% PEG. Collected fractions were monitored at 280 nm

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques: Conjugation Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: Conjugated Herceptin (homophilic Herceptin) at different amounts, 3.4 and 340 μg, was chromatographed on Sephacryl S300 in PBS. Overlay chromatograms with 3.4 and 340 μg, monitored with Ig-capture ELISA

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: Native gel electrophoresis with a non-denaturing detergent as described. Left lane 3.4 μg Homophilic Herceptin, right lane 3.4 μg of Herceptin

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques: Nucleic Acid Electrophoresis

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: Induction of apoptosis in H1650 cells using 2 μg/ml of homophilic Herceptin (a) or Herceptin (b)

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: Comparison of FACS intensity using Herceptin and homophilic Herceptin on live NSCLC H1650 cells. Solid and the dashed lines show Herceptin- and homophilic Herceptin-treated cells, respectively. a 0.05 μg/ml, b 0.1 μg/ml, c 0.5 μg/ml, d 1 μg/ml antibody

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques: Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: Comparison of FACS staining with homophilic Herceptin on fixed and live H1650 cells (dashed line and solid line, respectively). a FACS using secondary FITC antibody only. b Using 1 μg/ml of homophilic antibody. c Using 5 μg/ml of homophilic antibody

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques: Comparison, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: Comparison of FACS staining of live H1650 cells at 4 and 37°C [open circle (cold) and solid circle (warm), respectively]. a, b FACS gated at low-fluorescence intensity a using dilutions of homophilic Herceptin, and b dilutions of Herceptin. c, d FACS gated at high-fluorescence intensity c using dilutions of homophilic Herceptin, and d dilutions of Herceptin

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques: Comparison, Staining, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: FACS of live H1650 cells using untreated homophilic Herceptin or treated with gluteraldehyde (solid line and dotted line, respectively). a 1 μg/ml, b 5 μg/ml, and c 10 μg/ml

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: Induction of apoptosis in H1650 cells using different concentrations of homophilic Herceptin. H1650 cells were incubated overnight at 37°C with either no antibody or homophilic Herceptin. Cells were stained with Annexin V and PI, and analyzed on FACS. a No antibody. b 5 μg/ml antibody. c 10 μg/ml antibody. d 20 μg/ml antibody

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques: Incubation, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

doi: 10.1007/s00262-008-0597-z

Figure Lengend Snippet: Xenograft using H1650 cells in nude mice. 5 × 106 H1650 cells were injected into the flank of each mouse. Groups (12 mice each) were untreated (diamond), treated with Herceptin (solid box), or were treated with homophilic Herceptin (open box). Tumor growth was monitored three times weekly using a caliper and is shown on the y axis as cm3. Mice were treated twice weekly with antibody (110°μg/mouse) starting 24 h post-tumor inoculation. All groups of mice were euthanized at day 32. Error bars indicate the standard deviation of each group of 12 mice

Article Snippet: A 26 mer homophilic peptide [ 10 ], which included the C-terminal Ala-Trp homophilic domain, was made by AnaSpec, Inc (San Jose, CA).

Techniques: Injection, Standard Deviation