Journal: bioRxiv

Article Title: Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

doi: 10.1101/2025.09.16.676439

Figure Lengend Snippet: (a) Flow cytometry of fresh whole-blood samples from anonymous donors. C4 protein was quantified (Median Fluorescent Intensity) in major immune cell types using flow cytometry. Jurkat cell line is the negative control and hepG2 is the positive control cell line (top 2 panels in dark gray). Rabbit IgG isotype controls for each major immune cell type are shown in the panel below the C4 protein quantification in that immune cell type (light gray). The bottom panel shows FMO control (dark gray). (b) Median Fluorescence Intensity (MFI) in major immune cell types in ten healthy donors from the Stanford Blood Center, as measured by flow cytometry. Group differences between immune cell types were tested using the nonparametric Mann-Whitney U Test and significance was tested using the false discovery rate (FDR). * = p <0.05, ** = p <0.01, *** = p <0.001. (c) Comparisons that survived threshold for multiple comparisons are shown. A comprehensive list of comparisons is provided in Supplementary Table 3. (f) Average of Median Fluorescence Intensity (MFI) of C4 protein signal in major immune cell types in ten healthy donors from the Stanford Blood Center. An isotype control from the rabbit IgG isotype control for each immune cell type was also provided.

Article Snippet: Then, cells were resuspended in permeabilization buffer and an antibody against the C4 protein beta chain with a conjugated fluorescent tag (Complement C4 polyclonal antibody, AbBy Fluor 647 Conjugated, Bioss, Woburn, MA) for 30 minutes at room temperature with rotation in the dark.

Techniques: Flow Cytometry, Negative Control, Positive Control, Control, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

doi: 10.1101/2025.09.16.676439

Figure Lengend Snippet: (a) Flow cytometry was used to quantify C4 protein in major immune cell subtypes. (b) C4 protein was measured by immunoblotting against C4 protein (22233 Proteintech, Rosemont IL) using WES (capillary-based western blotting) in isolated neutrophils sampled from 15 SCZ and 21 control individuals. Samples were normalized using cellular actin (8H10D10, Invitrogen, Waltham, MA, USA). (c) Table showing the Descriptive statistics of measured immune cell-associated C4 protein in samples from individuals with SCZ compared to controls using different methods. Group comparisons of C4 protein for each major immune cell type (and plasma) were performed using one-way ANOVA. (d-f) CM and NCM were isolated from frozen live PBMC samples. Isolated cells were incubated, fixed, and stained for C4 protein (antibody directed against C4 protein, Proteintech, 22233-1-AP). C4 protein is localized throughout monocyte cells but is preferentially localized at the periphery. Representative images of CMs stained with Hoeschet stain for DNA and fluorescently labeled antibody against C4 protein in a sample from a control participant and a sample from a participant with SCZ. (e) Quantification of C4 protein throughout each cell was performed by measuring the Mean Intensity (Mean) from immunofluorescent images of C4 protein using Fiji. A mask was created from the nuclear stain and used to subtract the central C4 protein fluorescence to determine the Peripheral C4 protein Mean Fluorescence. (f) Table showing the exploratory descriptive statistics of CM and NCM C4 protein in samples from individuals with SCZ compared to controls. RFU = Relative Fluorescent Unit.

Article Snippet: Then, cells were resuspended in permeabilization buffer and an antibody against the C4 protein beta chain with a conjugated fluorescent tag (Complement C4 polyclonal antibody, AbBy Fluor 647 Conjugated, Bioss, Woburn, MA) for 30 minutes at room temperature with rotation in the dark.

Techniques: Flow Cytometry, Western Blot, Isolation, Control, Clinical Proteomics, Incubation, Staining, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1101/598342

Figure Lengend Snippet: (A) Diagram depicting IUE surgery performed in E16 dams. (B) Representative 20X confocal image of IUE with GFP targeted to L2/3 mPFC. Yellow asterisk: L2/3 GFP+ neurons. Left panel scale bar = 250 μm. Right panel scale bar = 75 μm. (C) Representative 60X confocal images of in situ hybridization from the same coronal section showing CaMKIIα+ neurons that are transfected with GFP and mC4 (C4B) (white arrowhead) and untransfected neighbors expressing mC4 (pink arrowhead) in P21 mice. Scale bar = 15 μm. (D) IUE reliably increases C4B transcript level in transfected cells. Percent of soma area positive for C4 transcript in transfected and untransfected neurons. N = 100 neurons (3 mice) per condition. t-test. **** p < 0.0001. Mean and SEM. (E) Transcript levels of GFP and mC4 positively correlate in transfected cells. Black line: linear fit. Gray lines: 95% confidence intervals. Black dotted line: average endogenous C4 expression in CaMKIIα+ mPFC L2/3 neurons. N = 100 transfected neurons (3 mice). Pearson’s R correlation and linear regression. R 2 = 0.28. **** p < 0.0001.

Article Snippet: DNA sequences containing mouse C4B (NM_009780.2, synthesized by Genescript) and human C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: In Situ Hybridization, Transfection, Expressing

Journal: bioRxiv

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1101/598342

Figure Lengend Snippet: (A) Developmental time course reveals significant decrease of spine density in neurons overexpressing C4 at P21-23. ** p < 0.01, **** p < 0.0001. (B) Representative 40X confocal images of P21-23 apical dendritic tufts. Scale bar = 5 μm. (C) Representative 40X confocal images of P21-23 apical dendritic spine types. Yellow asterisk: large mushroom spine (TIB (a.u.) > 75%). Green arrowhead: thin spine/filopodia (TIB (a.u.) < 25%). Scale bar = 3 μm. (D) Spine density sorted by spine types (spine/μm) reveals a specific reduction of medium-sized and thin/filopodia spine types in the mC4 condition. ** p < 0.01. (E) Representative 40X confocal images of P21-23 basal dendritic spines. Scale bar = 5 μm. (F) Analysis of basilar spine density (spine/μm) reveals no difference across groups. (A-F) N = 10-12 dendrites (from 4 mice) for each condition. Two-way ANOVA and Tukey’s test. Control: blue, mC4: red, hC4: purple. Mean and SEM.

Article Snippet: DNA sequences containing mouse C4B (NM_009780.2, synthesized by Genescript) and human C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques:

Journal: Proteomics. Clinical applications

Article Title: Use of a Targeted Urine Proteome Assay (TUPA) to Identify Protein Biomarkers of Delayed Recovery After Kidney Transplant

doi: 10.1002/prca.201600132

Figure Lengend Snippet: Relative Expression of 107 Urine Proteins in Kidney Transplant Patients as Determined by TUPA MRM Analyses 1

Article Snippet: ELISA Assays C4b-binding Protein Alpha Chain (C4BPA) was quantified with sandwich ELISA kit EC2202-1 from Assaypro.

Techniques: Expressing

Journal: Proteomics. Clinical applications

Article Title: Use of a Targeted Urine Proteome Assay (TUPA) to Identify Protein Biomarkers of Delayed Recovery After Kidney Transplant

doi: 10.1002/prca.201600132

Figure Lengend Snippet: Trajectory of Fold-Changes for the Top 4 Proteins. The fold-changes for the top 4 proteins, C4b-binding protein alpha chain (C4BPA), Serum amyloid P-component (SAMP), Immunoglobulin superfamily member 8 (IGSF8), and Guanylin (GUC2A) were determined by TUPA analyses described in the text and summarized in Table 1.

Article Snippet: ELISA Assays C4b-binding Protein Alpha Chain (C4BPA) was quantified with sandwich ELISA kit EC2202-1 from Assaypro.

Techniques: Binding Assay

Journal: Proteomics. Clinical applications

Article Title: Use of a Targeted Urine Proteome Assay (TUPA) to Identify Protein Biomarkers of Delayed Recovery After Kidney Transplant

doi: 10.1002/prca.201600132

Figure Lengend Snippet: Average ELISA and MRM Fold-Changes for C4b-binding Protein Alpha Chain and Serum Amyloid P-Component

Article Snippet: ELISA Assays C4b-binding Protein Alpha Chain (C4BPA) was quantified with sandwich ELISA kit EC2202-1 from Assaypro.

Techniques: Enzyme-linked Immunosorbent Assay