Journal: bioRxiv

Article Title: Identifying membrane-bound transcriptional regulatory proteins from rare but evolutionarily conserved domain combinations

doi: 10.64898/2025.12.20.695554

Figure Lengend Snippet: Based on their conserved domain compositions, PREB, CERS2, and NAT8F3 are new candidate MBTRs ( a ) AlphaFold-predicted structures for three potential MBTRs, here showing the mouse proteins. Transmembrane domains are marked in yellow. The domains that comprise the combination with the transmembrane domain are colored blue. ( b ) Phylogenetic trees showing 10 orthologs of each candidate protein with their Pfam domain annotations (top: PREB, middle: CERS2, bottom: NAT8F3 and NAT8F7). The TLC domain includes internal transmembrane domains. The same color scheme as panel ( a ) was applied.

Article Snippet: Mouse Preb cDNA ORF clones were obtained from Sino Biological (MG5A1071-CY) and amplified using primers designed to produce constructs encoding PREB with or without the transmembrane domain (PREBΔTM) ( Supplementary Table S1 ), with the incorporation of Notl and NheI restriction sites (NEB, R3189 and R3131).

Techniques:

Journal: bioRxiv

Article Title: Identifying membrane-bound transcriptional regulatory proteins from rare but evolutionarily conserved domain combinations

doi: 10.64898/2025.12.20.695554

Figure Lengend Snippet: PREB without its transmembrane domain undergoes nuclear translocation and binds to the butyrophilin locus. ( a ) Predicted structure of PREB generated by AlphaFold, highlighting the WD repeat domains, the transmembrane domain, and the site of an engineered C-terminal truncation. ( b ) Schematic of the two constructs created to assay the mouse full-length PREB protein and PREBΔTM, lacking the C-terminal transmembrane domain. The N-terminal biotinylation site is indicated. ( c ) Western blot results of two biological replicates of ES cells expressing PREB and PREBΔTM. Two bands for PREB were detected by Streptavidin-HRP, suggesting C-terminal processing of a full-length PREB when expressed in mouse ES cells. ( d ) Immunofluorescence confocal microscopy images of PREB and PREBΔTM following Dox induction. PREB localizes predominantly to a perinuclear region, forming a ring-like shape. PREBΔTM displays diffused nuclear localization. Rightmost panels show zoomed images corresponding to the boxed regions. ( e ) bioChIP-seq profiles for PREB, PREBΔTM, BirA-only Strep-pulldown, and IgG ChIP-seq control samples. PREBΔTM samples show substantially stronger DNA-binding activity than full-length PREB. ( f ) Representative locus-specific binding profiles of PREB and PREBΔTM at the Btn1a1 (butyrophilin subfamily 1 member A1) gene. PREBΔTM shows markedly higher peak intensities, consistent with enhanced nuclear localization in the absence of the transmembrane domain.

Article Snippet: Mouse Preb cDNA ORF clones were obtained from Sino Biological (MG5A1071-CY) and amplified using primers designed to produce constructs encoding PREB with or without the transmembrane domain (PREBΔTM) ( Supplementary Table S1 ), with the incorporation of Notl and NheI restriction sites (NEB, R3189 and R3131).

Techniques: Translocation Assay, Generated, Construct, Western Blot, Expressing, Immunofluorescence, Confocal Microscopy, ChIP-sequencing, Control, Binding Assay, Activity Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: scRNA-seq reveals irradiation-induced Trem2 expression in skin macrophages. (A) tSNE plots showing cell type annotation based on CellMarker 2.0, and distribution of cell clusters in control and irradiated groups. (B) Proportion of each cell population comparing control and irradiation groups. (C) Annotation of macrophage-related clusters using marker genes. (D) Quantification of macrophage cluster ratios between control and irradiated groups. (E) Dotplot showing the expression of representative genes in macrophage subclusters, highlighting Trem2 -specific enrichment. (F) irGSEA of macrophage clusters, including a gene expression heatmap and GSEA plot. (G) AUCell analysis indicating high activity of the PI3K-Akt pathway in MAC2 macrophage subclusters. (H) Separation of macrophage clusters based on Trem2 expression. (I) Comparison of the ratio of Trem2 -positive and Trem2 -negative clusters between control and irradiated samples. (J) Pseudotime trajectory analysis illustrating the developmental progression of Trem2 -positive and Trem2-negative macrophages.

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Irradiation, Expressing, Control, Marker, Gene Expression, Activity Assay, Comparison

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Radiation-induced ROS–Nrf2–ADAM17 axis promotes Trem2 shedding. (A) Quantification of sTREM2 in culture supernatants of RAW264.7 cells under control and irradiated conditions ( n = 3). (B) ADAM10 transcript levels from RNA-seq and relative mRNA expression measured by qPCR ( n = 3). (C) ADAM17 transcript levels from RNA-seq and relative mRNA expression measured by qPCR ( n = 3). (D and E) Western blotting and analysis of ADAM10 and ADAM17 ( n = 3). (F) Pearson correlation analysis between Trem2 , ADAM10 , ADAM17 , and Nrf2 based on RNA-seq data. (G) GSEA showing enrichment of the “Positive Regulation of Reactive Oxygen Species Metabolic Process” pathway after irradiation. (H) (a) ROS levels detected by fluorescence staining at 2 and 6 h post-irradiation (green), shown with brightfield (BF) (scale bar, 200 μm); (b) quantification of integrated fluorescence intensity (IntDen) ( n = 3). (I) ROS quantification based on fluorescence intensity at excitation/emission wavelengths of 488/525 nm ( n = 3). (J) (a and b) IF detection and quantification of NRF2 (green) with DAPI staining ( n = 3; scale bar, 200 μm); (c and d) Western blotting and quantification of NRF2 ( n = 3). (K and L) Western blotting and quantification of TREM2, BAX, and BCL2 protein levels in control, DMSO, and GW280264X cells with or without irradiation ( n = 3). (M) ELISA detection of sTREM2 in various treatment groups ( n = 3). (N) Cell viability assay using CCK-8 in control (Ctr), DMSO, and GW280264X (ADAM17/ADAM10 inhibitor) groups with or without irradiation ( n = 3). (O) ELISA detection of proinflammatory cytokines IL-1β, TNF-α, and IL-6 in various treatment groups ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Control, Irradiation, RNA Sequencing, Expressing, Western Blot, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Viability Assay, CCK-8 Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Trem2 overexpression improves radiation resistance of RAW264.7 cells by reducing apoptosis. (A and B) Live/dead staining using Calcein-AM (live, green) and EthD-1 (dead, red); quantification of dead cells based on integrated fluorescence intensity ( n = 3; scale bar, 200 μm). (C) Cell viability assessed by CCK-8 assay in Blank, Vector, OE-Trem2, siNC, and si-Trem2 groups with or without irradiation ( n = 3). (D and E) Scratch assay and distance closure rate quantified at 6, 12, and 24 h relative to 0 h ( n = 3; scale bar, 200 μm). (F) Apoptosis rates determined by flow cytometry across different treatment groups ( n = 3). (G to J) Western blotting and analysis of CD206, CD86, CASPASE-9 (CAS9), total and cleaved CASPASE-3 (pro-CAS3, cleaved CAS3), BCL2, BAX, and cleaved CASPASE-8 (cleaved CAS8) ( n = 3). (K) ELISA analysis of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and the anti-inflammatory cytokine IL-10 in different groups ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; only statistically significant comparisons are shown in panel.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Over Expression, Staining, Fluorescence, CCK-8 Assay, Plasmid Preparation, Irradiation, Wound Healing Assay, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Trem2 deficiency exacerbates radiation-induced apoptosis by suppressing ERK phosphorylation. (A and B) Western blotting and quantification of TREM2 expression in LysM Cr e Trem2 flox/flox (Trem2-Cko) and LysM − Trem2 flox/flox (Trem2-Ctr) BMDM, with or without irradiation ( n = 3). (C) Flow cytometry analysis of macrophage polarization based on M1 markers (CD86 + CD206 − ) and M2 markers (CD86 − CD206 + ) ( n = 3). (D) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis based on RNA-seq data. (E) Apoptosis rate assessed by Annexin V/PI flow cytometry in Trem2-Ctr and Trem2-Cko macrophages after irradiation ( n = 3). (F and G) Western blotting and analysis of phosphorylated ERK1/2 (p-ERK), total ERK1/2 (t-ERK), CASPASE-9 (CAS9), total and cleaved CASPASE-3 (pro-CAS3, cleaved CAS3), BCL2, BAX, and cleaved CASPASE-8 (cleaved CAS8) ( n = 3). (H) ELISA quantification of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and anti-inflammatory cytokine IL-10 in Trem2-Ctr and Trem2-Cko groups ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Phospho-proteomics, Western Blot, Expressing, Irradiation, Flow Cytometry, RNA Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Radiation delayed wound healing in radiation-induced skin injury (RISI) and induced TREM2 expression. (A) The RISI mouse model: WT mice received 5-Gy total body x-ray irradiation followed by full-thickness excisional skin wounding (black circle, 12-mm inner diameter). (B) Representative wound area measurements ( n = 6). (C) Quantification of wound closure rates at various time points relative to post-injury day 0 (PID0) ( n = 6). (D and E) H&E and Masson staining of wound tissue sections at PID0, 3, 7, and 14, showing regions close to the wound margin; IF costaining of TREM2 (gold), F4/80 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) to identify TREM2 + macrophages in control and irradiated groups (scale bar was shown on panel). (F) Quantification of collagen deposition based on Masson staining ( n = 6). (G) Total macrophage ratio calculated as the proportion of F4/80 + DAPI + cells among total DAPI + cells by immunohistochemistry ( n = 6). (H) TREM2 + macrophage ratio calculated as the proportion of TREM2 + F4/80 + DAPI + cells among total F4/80 + DAPI + macrophages ( n = 6). (I) qPCR analysis of Trem2 mRNA expression at different time points ( n = 6). For within-group comparisons, expression was normalized to day 0 of each group. For between-group comparisons, expression levels in the irradiation group were normalized to the corresponding control group at each time point ( n = 6). (J and K) Western blotting and analysis of TREM2 protein expression across time points in control and irradiated groups ( n = 6). (L) ELISA measurement of soluble TREM2 (sTREM2) levels in skin tissue at different time points ( n = 6). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Expressing, Irradiation, Staining, Control, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Radiation reduced RAW264.7 cell viability and induced Trem2 expression. (A) Volcano plot showing DEGs between irradiation and control RAW264.7 cells based on RNA-seq ( n = 3). (B) GSEA identifying Trem2 -associated pathways enriched or suppressed following irradiation. (C) Heatmap of representative genes involved in macrophage differentiation, proliferation, migration, and apoptosis from GSEA-identified pathways. Significantly up-regulated and down-regulated genes are marked with upward (↑) and downward (↓) arrows, respectively. (D and E) Flow cytometry analysis of apoptosis using Annexin V and propidium iodide (PI) staining. Early and late apoptotic cells were quantified from Q2 and Q3 quadrants ( n = 3). (F and G) Live (green)/dead (red) staining; dead cell proportion calculated based on integrated fluorescence density ( n = 3; scale bar, 200 μm). (H) CCK-8 assay shown relative cell viability at 24 h post-irradiation compared to 0 h ( n = 3 biological replicates with 3 technical repeats each). (I) Trem2 transcript level from RNA-seq data. (J) qPCR analysis of Trem2 mRNA expression across different treatment groups ( n = 3). (K and L) Western blot and analysis of TREM2, BAX, and BCL2 protein expression ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Expressing, Irradiation, Control, RNA Sequencing, Migration, Flow Cytometry, Staining, Fluorescence, CCK-8 Assay, Western Blot

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Trem2 deficiency impairs wound healing in RISI. (A) RISI mouse model: Trem2-Ctr and Trem2-Cko mice were subjected to 5-Gy total body x-ray irradiation followed by full-thickness excisional wounding (black circle, 12-mm inner diameter). (B) Representative images of wound areas in each group ( n = 6). (C) Quantification of wound closure rates at indicated time points relative to PID0 ( n = 6). (D) H&E and Masson staining of wound tissues at PID0, 3, 7, and 14 ( n = 6). (E) IF costaining of iNOS (red), CD206 (green), and DAPI (blue) to identify M1- or M2-type macrophages in Trem2-Ctr and Trem2-Cko groups (scale bars shown in panels). (F) Collagen deposition quantified from Masson staining ( n = 6). (G) Quantification of macrophage subsets, calculated as the proportion of iNOS + CD206 − DAPI + cells (M1) or iNOS − CD206 + DAPI + cells (M2) among total DAPI + cells ( n = 6). (H) ELISA quantification of proinflammatory cytokines (IL-1β, TNF-α, and IL-6), anti-inflammatory cytokine IL-10, and total CASPASE3 in Trem2-Ctr and Trem2-Cko groups ( n = 6).

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Irradiation, Staining, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Schematic of TREM2 regulates macrophage apoptosis and impairs wound healing following RISI.

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques:

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Schematic of PEG-Maleimide (PEG-Mal) assay for assaying disulfide bond formation in recombinant mouse PrP (recMoPrP). ( b ) recMoPrP was reduced with tris(2-carboxyethyl)phosphine (TCEP), treated with (+) or without (-) recombinant P4HB (recP4HB), and then proteins were labeled with PEG-Mal. PEG-Mal labeled proteins were resolved by SDS-PAGE and analyzed by immunoblotting. The blot was probed with the anti-PrP antibody SAF32 or an anti-P4HB antibody. ( c ) Quantification of the relative ratio of PEG-Mal-labeled to unlabeled PrP for reactions performed in the absence (-) and presence (+) of recP4HB. Data are mean ± SEM from four independent replicates. Statistical significance was assessed using an unpaired, two-tailed t test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Recombinant, Labeling, SDS Page, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots for PrP C , P4HB, GRP78, PDIA3 and PDIA4 in cell lysates from CAD5 cells transfected with control or P4HB siRNA for 72 h. ( b, c ) Quantification of PrP C levels (b) and PrP C glycoforms (c) in CAD5 cells transfected with control or P4HB siRNA (n = 6). ( d ) Quantification of P4HB, GRP78, PDIA3, and PDIA4 levels in CAD5 cells transfected with control or P4HB siRNA (n = 4). ( e ) Immunofluorescence images of CAD5 cells transfected with control or P4HB siRNA. PrP C (green) and P4HB (red) were revealed using the anti-PrP antibody POM1 and an anti-P4HB antibody, and nuclei were stained with DAPI (blue). Scale bar = 10 μm (applies to all images). ( f ) Immunoblots of detergent-insoluble PrP and P4HB species in CAD5 cells transfected with control or P4HB siRNA for 72 h. ( g ) Quantification of detergent-insoluble PrP levels in CAD5 cells transfected with control or P4HB siRNA (n = 4). ( h ) Immunoblot of proteinase K (PK)-resistant PrP (PrP res ) levels in 22L prion-infected CAD5 cells transfected with control or P4HB siRNA for 72 h. ( i, j ) Quantification of PrP res levels (i) and PrP res glycoforms (j) in 22L prion-infected CAD5 cells transfected with control or P4HB siRNA (n = 8). In panels b, c, d, g, i, and j, data are mean ± SEM and statistical significance was assessed using unpaired, two-tailed t tests.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Transfection, Control, Immunofluorescence, Staining, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Immunoblot of secreted PrP levels in conditioned medium from CAD5 cells transfected with control or P4HB siRNA. The membrane was stained for total protein following transfer using Ponceau S. ( b ) Quantification of secreted PrP levels in CAD5 cells transfected with control or P4HB siRNA (n = 4 independent replicates). Data is mean ± SEM and statistical significance was assessed using an unpaired two-tailed t test. ( c ) Immunoblot of secreted PrP levels in conditioned medium from CAD5 cells stably transfected with control or one of two distinct P4HB shRNAs. The membrane was stained for total protein following transfer using Ponceau S. ( d ) Quantification of secreted PrP levels in CAD5 cells stably transfected with control or P4HB shRNA (n = 6 independent replicates). Data is mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Transfection, Control, Membrane, Staining, Two Tailed Test, Stable Transfection, shRNA

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Immunoblot of proteinase K (PK)-resistant PrP (PrP res ) levels in RML prion-infected CAD5 cells transfected with control or P4HB siRNA for 72 h. ( b ) Quantification of PrP res levels in RML prion-infected CAD5 cells transfected with control or P4HB siRNA (n = 8 independent replicates). Data are mean ± SEM and statistical significance was assessed using an unpaired two-tailed t test. ( c ) Quantification of PrP res glycoforms in RML prion-infected CAD5 cells transfected with control or P4HB siRNA (n = 8 independent replicates). Data are mean ± SEM and statistical significance was assessed using an unpaired two-tailed t tests.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Infection, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots for PrP C , P4HB, GRP78, PDIA3 and PDIA4 in cell lysates from CAD5 cells stably transfected with control or one of two distinct P4HB shRNAs. ( b, c ) Quantification of PrP C and P4HB levels (b) as well as GRP78, PDIA3, and PDIA4 levels (c) in CAD5 cells stably transfected with control or P4HB shRNAs (n = 5). ( d ) Immunofluorescence images of CAD5 cells stably transfected with control or P4HB shRNAs. PrP C (green) and P4HB (red) were revealed using the anti-PrP antibody POM1 and an anti-P4HB antibody, and nuclei were stained with DAPI (blue). Scale bar = 10 μm (applies to all images). ( e ) Immunoblots of detergent-insoluble PrP and P4HB species in CAD5 cells stably transfected with control or P4HB shRNAs. ( f ) Quantification of the ratio of insoluble:total PrP in CAD5 cells transfected with control or P4HB shRNA (n = 4). ( g, i ) Immunoblots of PrP res levels in CAD5 cells stably transfected with control or P4HB shRNAs and then infected with either RML (g) or 22L (i) prions. ( h, j ) Quantification of PrP res levels in CAD5 cells stably transfected with control or P4HB shRNAs and then infected with either RML (h) or 22L (j) prions (n = 8). In panels b, c, f, h, and j, data are mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Stable Transfection, Transfection, Control, Immunofluorescence, Staining, shRNA, Infection

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Schematic structure of P4HB, which features catalytic a and a′ domains (orange), non-catalytic ligand-binding domains b and b′ (green), and an ER-retention motif (KDEL) at the C-terminus. The selective P4HB inhibitor, KSC-34, is 30-fold more selective for the a site than the a′ site with an IC 50 of ∼3.5 μM. ( b ) Lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h were labeled with or without PEG-PCMal, and then PEG-PCMal labeled proteins were examined by immunoblotting. The blot was probed with antibodies to P4HB, PDIA3, and PDIA4. ( c ) Representative immunoblots for PrP C , P4HB, GRP78, PDIA3, and PDIA4 in cell lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. ( d, e ) Quantification of PrP C (d) as well as P4HB, GRP78, PDIA3, and PDIA4 (e) levels in CAD5 cells treated with 0 or 3.5 µM KSC-34 for 72 h (n = 4). ( f ) Quantification of PrP C glycoforms in CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h (n = 4). ( g ) Immunofluorescence images of CAD5 cells treated with 0 or 3.5 µM KSC-34 for 72 h. The expression of PrP C (green) and P4HB (red) were revealed using the anti-PrP antibody POM1 and an anti-P4HB antibody, and nuclei were stained with DAPI (blue). Scale bar = 10 μm (applies to all images). ( h ) Representative immunoblots of PrP C , P4HB, and GRP78 in cell lysates from CAD5 cells treated for 1, 2, or 3 days with 0 or 3.5 µM KSC-34. ( i ) Quantification of PrP C levels in CAD5 cells treated for 1, 2, or 3 days with 0 or 3.5 µM KSC-34 (n = 4). ( j ) Representative immunoblots for detergent-insoluble PrP and P4HB species in cell lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. ( k ) Quantification of detergent-insoluble PrP and P4HB levels in CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h (n = 4). ( l ) Quantification of the ratio of insoluble:total PrP in CAD5 cells treated with 0 or 3.5 µM KSC-34 for 72 h (n = 4). All data are mean ± SEM. In panels d, e, k, and l, statistical significance was assessed using unpaired two-tailed t tests. In panels f and i, statistical significance was assessed using a two-way ANOVA followed by Šídák’s multiple comparison test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Ligand Binding Assay, Labeling, Western Blot, Immunofluorescence, Expressing, Staining, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: Representative immunoblots for PrP C and P4HB in cell lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. In reduced conditions, lysates were treated with β-mercaptoethanol and boiled. PrP C was detected using the antibodies D18 or POM2.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots of PrP C , P4HB, GRP78, and actin levels in differentiated CAD5 cells treated with 0 or 3.5 µM KSC-34 for 1, 2, or 3 days. ( b ) Quantification of PrP C levels in differentiated CAD5 cells treated with 0 or 3.5 µM KSC-34 for 1, 2, or 3 days. The graph displays mean ± SEM and statistical significance was assessed using two-way ANOVA followed by Šídák’s multiple comparison test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Comparison

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots of total PrP (-PK), PrP res (+PK), P4HB, and actin levels in cell lysates from mock-infected (Ctrl) CAD5 cells and CAD5 cells that have been stably infected with either RML or 22L prions. ( b ) Quantification of P4HB levels in cell lysates from Ctrl-infected CAD5 cells and CAD5 cells that have been stably infected with either RML or 22L prions. Data is mean ± SEM from 4 independent replicates. Statistical significance was assessed using unpaired two-tailed t tests.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Infection, Stable Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots for total PrP, P4HB and proteinase K (PK)-resistant PrP Sc (PrP res ) levels in lysates from either RML or 22L prion-infected CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. The blots were probed with the anti-PrP antibody D13, an anti-P4HB antibody, and an anti-actin antibody. ( b-d ) Quantification of total PrP (b), PrP res (c), and P4HB (d) levels in RML or 22L prion-infected CAD5 cells treated with 0 or 3.5 μM KSC-34 (n = 4 independent replicates). ( e ) Representative immunoblot of PrP res levels in RML prion-infected CAD5 cells treated with 0, 3.5, or 7 µM KSC-34 for 72 h. ( f ) Quantification of PrP res levels in RML prion-infected CAD5 cells treated with 0, or 3.5, or 7 µM KSC-34 (n = 4 independent replicates). ( g ) Representative immunoblot of PrP res levels in RML prion-infected CAD5 cells cultured in the continuous presence of either 0 or 3.5 µM KSC-34 for 4 passages. ( h ) Quantification of PrP res levels in RML prion-infected CAD5 cells treated with 0 or 3.5 µM KSC-34 for X days (n = 6 independent replicates). In panels b-d, f, and h, graphs display mean ± SEM. Statistical significance in panels b-d, and h was assessed using unpaired, two-tailed t tests. In panel f, statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Infection, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Immunoblots for PrP C and P4HB in total cell lysates as well as unbound and eluate fractions following incubation with streptavidin agarose from CAD5 cells subjected to cell surface biotinylation (+) or mock (-) treatment. ( b ) Schematic structures of WT mouse P4HB as well as mutants with a catalytically inactive a site (C55/58S) and/or deletion of the C-terminal ER retention signal (ΔKDEL). ( c ) Immunoblot of PrP C , P4HB, and actin levels in cell lysates from CAD5 cells transiently transfected with empty vector (EV) or with WT or mutant P4HB for 48 h. ( d ) Quantification of PrP C and P4HB levels in lysates from transiently transfected CAD5 cells (n = 4 independent replicates). ( e ) Immunoblot of secreted PrP and P4HB levels in the conditioned medium from CAD5 cells transiently transfected with EV or with WT or mutant P4HB for 48 h. As a control, the membrane was stained for total protein following transfer using Ponceau S. ( f ) Representative immunoblots for proteinase K (PK)-resistant PrP Sc (PrP res ) levels in lysates from either RML or 22L prion-infected CAD5 cells transiently transfected with EV or with WT or mutant P4HB for 48 h. ( g ) Quantification of PrP res levels in transiently transfected RML or 22L prion-infected CAD5 cells (n = 4 independent replicates). In panels d and g, graphs display mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Incubation, Transfection, Plasmid Preparation, Mutagenesis, Control, Membrane, Staining, Infection

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Immunoblot of secreted P4HB levels in the conditioned medium (CM) from CAD5-PrP -/- cells transiently transfected with empty vector (EV) or with mutant P4HB variants lacking the C-terminal ER retention signal. As a control, the membrane was stained for total protein following transfer using Ponceau S. ( b ) Schematic of the experiment in which CM from transiently transfected CAD5-PrP -/- cells is applied to 22L prion-infected CAD5 cells for 72 h. ( c ) Representative immunoblot of proteinase K (PK)-resistant PrP Sc (PrP res ) levels in lysates from 22L prion-infected CAD5 cells treated with CM from CAD5-PrP -/- cells transiently transfected with EV or secreted P4HB variants. ( d ) Quantification of PrP res levels in lysates from 22L prion-infected CAD5 cells treated with CM from CAD5-PrP -/- cells transiently transfected with EV or secreted P4HB variants (n = 7 independent replicates). The graph displays mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Control, Membrane, Staining, Infection

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Schematic of the experiment in which conditioned medium (CM) from transiently transfected CAD5-PrP -/- cells is applied to RML prion-infected CAD5 cells for 72 h. ( b ) Representative immunoblot of proteinase K (PK)-resistant PrP Sc (PrP res ) levels in lysates from RML prion-infected CAD5 cells treated with CM from CAD5-PrP -/- cells transiently transfected with empty vector (EV) or secreted P4HB variants. ( d ) Quantification of PrP res levels in lysates from RML prion-infected CAD5 cells treated with CM from CAD5-PrP -/- cells transiently transfected with EV or secreted P4HB variants (n = 7 independent replicates). The graph displays mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Transfection, Infection, Western Blot, Plasmid Preparation

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) P4HB helps to maintain the disulfide bond in PrP C . When P4HB is depleted or its disulfide isomerase activity is inhibited, the disulfide bond in PrP C forms less efficiently causing PrP C to become misfolded and degraded, which leads to lower steady-state PrP C levels. ( b ) P4HB functions as a chaperone that potentiates prion replication. In prion-infected cells, P4HB may act as a scaffold that facilitates interactions between PrP C and PrP Sc . ( c ) When P4HB levels are decreased, both the isomerase and chaperone activities of P4HB are decreased, leading to lower PrP C levels and less prion replication. When the catalytic a domain is inhibited, the chaperone activity of P4HB increases, leading to increased prion replication despite lower PrP C levels.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Activity Assay, Infection

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Schematic of PEG-Maleimide (PEG-Mal) assay for assaying disulfide bond formation in recombinant mouse PrP (recMoPrP). ( b ) recMoPrP was reduced with tris(2-carboxyethyl)phosphine (TCEP), treated with (+) or without (-) recombinant P4HB (recP4HB), and then proteins were labeled with PEG-Mal. PEG-Mal labeled proteins were resolved by SDS-PAGE and analyzed by immunoblotting. The blot was probed with the anti-PrP antibody SAF32 or an anti-P4HB antibody. ( c ) Quantification of the relative ratio of PEG-Mal-labeled to unlabeled PrP for reactions performed in the absence (-) and presence (+) of recP4HB. Data are mean ± SEM from four independent replicates. Statistical significance was assessed using an unpaired, two-tailed t test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Recombinant, Labeling, SDS Page, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots for PrP C , P4HB, GRP78, PDIA3 and PDIA4 in cell lysates from CAD5 cells transfected with control or P4HB siRNA for 72 h. ( b, c ) Quantification of PrP C levels (b) and PrP C glycoforms (c) in CAD5 cells transfected with control or P4HB siRNA (n = 6). ( d ) Quantification of P4HB, GRP78, PDIA3, and PDIA4 levels in CAD5 cells transfected with control or P4HB siRNA (n = 4). ( e ) Immunofluorescence images of CAD5 cells transfected with control or P4HB siRNA. PrP C (green) and P4HB (red) were revealed using the anti-PrP antibody POM1 and an anti-P4HB antibody, and nuclei were stained with DAPI (blue). Scale bar = 10 μm (applies to all images). ( f ) Immunoblots of detergent-insoluble PrP and P4HB species in CAD5 cells transfected with control or P4HB siRNA for 72 h. ( g ) Quantification of detergent-insoluble PrP levels in CAD5 cells transfected with control or P4HB siRNA (n = 4). ( h ) Immunoblot of proteinase K (PK)-resistant PrP (PrP res ) levels in 22L prion-infected CAD5 cells transfected with control or P4HB siRNA for 72 h. ( i, j ) Quantification of PrP res levels (i) and PrP res glycoforms (j) in 22L prion-infected CAD5 cells transfected with control or P4HB siRNA (n = 8). In panels b, c, d, g, i, and j, data are mean ± SEM and statistical significance was assessed using unpaired, two-tailed t tests.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Transfection, Control, Immunofluorescence, Staining, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Immunoblot of secreted PrP levels in conditioned medium from CAD5 cells transfected with control or P4HB siRNA. The membrane was stained for total protein following transfer using Ponceau S. ( b ) Quantification of secreted PrP levels in CAD5 cells transfected with control or P4HB siRNA (n = 4 independent replicates). Data is mean ± SEM and statistical significance was assessed using an unpaired two-tailed t test. ( c ) Immunoblot of secreted PrP levels in conditioned medium from CAD5 cells stably transfected with control or one of two distinct P4HB shRNAs. The membrane was stained for total protein following transfer using Ponceau S. ( d ) Quantification of secreted PrP levels in CAD5 cells stably transfected with control or P4HB shRNA (n = 6 independent replicates). Data is mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Transfection, Control, Membrane, Staining, Two Tailed Test, Stable Transfection, shRNA

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Immunoblot of proteinase K (PK)-resistant PrP (PrP res ) levels in RML prion-infected CAD5 cells transfected with control or P4HB siRNA for 72 h. ( b ) Quantification of PrP res levels in RML prion-infected CAD5 cells transfected with control or P4HB siRNA (n = 8 independent replicates). Data are mean ± SEM and statistical significance was assessed using an unpaired two-tailed t test. ( c ) Quantification of PrP res glycoforms in RML prion-infected CAD5 cells transfected with control or P4HB siRNA (n = 8 independent replicates). Data are mean ± SEM and statistical significance was assessed using an unpaired two-tailed t tests.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Infection, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots for PrP C , P4HB, GRP78, PDIA3 and PDIA4 in cell lysates from CAD5 cells stably transfected with control or one of two distinct P4HB shRNAs. ( b, c ) Quantification of PrP C and P4HB levels (b) as well as GRP78, PDIA3, and PDIA4 levels (c) in CAD5 cells stably transfected with control or P4HB shRNAs (n = 5). ( d ) Immunofluorescence images of CAD5 cells stably transfected with control or P4HB shRNAs. PrP C (green) and P4HB (red) were revealed using the anti-PrP antibody POM1 and an anti-P4HB antibody, and nuclei were stained with DAPI (blue). Scale bar = 10 μm (applies to all images). ( e ) Immunoblots of detergent-insoluble PrP and P4HB species in CAD5 cells stably transfected with control or P4HB shRNAs. ( f ) Quantification of the ratio of insoluble:total PrP in CAD5 cells transfected with control or P4HB shRNA (n = 4). ( g, i ) Immunoblots of PrP res levels in CAD5 cells stably transfected with control or P4HB shRNAs and then infected with either RML (g) or 22L (i) prions. ( h, j ) Quantification of PrP res levels in CAD5 cells stably transfected with control or P4HB shRNAs and then infected with either RML (h) or 22L (j) prions (n = 8). In panels b, c, f, h, and j, data are mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Stable Transfection, Transfection, Control, Immunofluorescence, Staining, shRNA, Infection

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Schematic structure of P4HB, which features catalytic a and a′ domains (orange), non-catalytic ligand-binding domains b and b′ (green), and an ER-retention motif (KDEL) at the C-terminus. The selective P4HB inhibitor, KSC-34, is 30-fold more selective for the a site than the a′ site with an IC 50 of ∼3.5 μM. ( b ) Lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h were labeled with or without PEG-PCMal, and then PEG-PCMal labeled proteins were examined by immunoblotting. The blot was probed with antibodies to P4HB, PDIA3, and PDIA4. ( c ) Representative immunoblots for PrP C , P4HB, GRP78, PDIA3, and PDIA4 in cell lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. ( d, e ) Quantification of PrP C (d) as well as P4HB, GRP78, PDIA3, and PDIA4 (e) levels in CAD5 cells treated with 0 or 3.5 µM KSC-34 for 72 h (n = 4). ( f ) Quantification of PrP C glycoforms in CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h (n = 4). ( g ) Immunofluorescence images of CAD5 cells treated with 0 or 3.5 µM KSC-34 for 72 h. The expression of PrP C (green) and P4HB (red) were revealed using the anti-PrP antibody POM1 and an anti-P4HB antibody, and nuclei were stained with DAPI (blue). Scale bar = 10 μm (applies to all images). ( h ) Representative immunoblots of PrP C , P4HB, and GRP78 in cell lysates from CAD5 cells treated for 1, 2, or 3 days with 0 or 3.5 µM KSC-34. ( i ) Quantification of PrP C levels in CAD5 cells treated for 1, 2, or 3 days with 0 or 3.5 µM KSC-34 (n = 4). ( j ) Representative immunoblots for detergent-insoluble PrP and P4HB species in cell lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. ( k ) Quantification of detergent-insoluble PrP and P4HB levels in CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h (n = 4). ( l ) Quantification of the ratio of insoluble:total PrP in CAD5 cells treated with 0 or 3.5 µM KSC-34 for 72 h (n = 4). All data are mean ± SEM. In panels d, e, k, and l, statistical significance was assessed using unpaired two-tailed t tests. In panels f and i, statistical significance was assessed using a two-way ANOVA followed by Šídák’s multiple comparison test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Ligand Binding Assay, Labeling, Western Blot, Immunofluorescence, Expressing, Staining, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: Representative immunoblots for PrP C and P4HB in cell lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. In reduced conditions, lysates were treated with β-mercaptoethanol and boiled. PrP C was detected using the antibodies D18 or POM2.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots of PrP C , P4HB, GRP78, and actin levels in differentiated CAD5 cells treated with 0 or 3.5 µM KSC-34 for 1, 2, or 3 days. ( b ) Quantification of PrP C levels in differentiated CAD5 cells treated with 0 or 3.5 µM KSC-34 for 1, 2, or 3 days. The graph displays mean ± SEM and statistical significance was assessed using two-way ANOVA followed by Šídák’s multiple comparison test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Comparison

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots of total PrP (-PK), PrP res (+PK), P4HB, and actin levels in cell lysates from mock-infected (Ctrl) CAD5 cells and CAD5 cells that have been stably infected with either RML or 22L prions. ( b ) Quantification of P4HB levels in cell lysates from Ctrl-infected CAD5 cells and CAD5 cells that have been stably infected with either RML or 22L prions. Data is mean ± SEM from 4 independent replicates. Statistical significance was assessed using unpaired two-tailed t tests.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Infection, Stable Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Representative immunoblots for total PrP, P4HB and proteinase K (PK)-resistant PrP Sc (PrP res ) levels in lysates from either RML or 22L prion-infected CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. The blots were probed with the anti-PrP antibody D13, an anti-P4HB antibody, and an anti-actin antibody. ( b-d ) Quantification of total PrP (b), PrP res (c), and P4HB (d) levels in RML or 22L prion-infected CAD5 cells treated with 0 or 3.5 μM KSC-34 (n = 4 independent replicates). ( e ) Representative immunoblot of PrP res levels in RML prion-infected CAD5 cells treated with 0, 3.5, or 7 µM KSC-34 for 72 h. ( f ) Quantification of PrP res levels in RML prion-infected CAD5 cells treated with 0, or 3.5, or 7 µM KSC-34 (n = 4 independent replicates). ( g ) Representative immunoblot of PrP res levels in RML prion-infected CAD5 cells cultured in the continuous presence of either 0 or 3.5 µM KSC-34 for 4 passages. ( h ) Quantification of PrP res levels in RML prion-infected CAD5 cells treated with 0 or 3.5 µM KSC-34 for X days (n = 6 independent replicates). In panels b-d, f, and h, graphs display mean ± SEM. Statistical significance in panels b-d, and h was assessed using unpaired, two-tailed t tests. In panel f, statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Infection, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Immunoblots for PrP C and P4HB in total cell lysates as well as unbound and eluate fractions following incubation with streptavidin agarose from CAD5 cells subjected to cell surface biotinylation (+) or mock (-) treatment. ( b ) Schematic structures of WT mouse P4HB as well as mutants with a catalytically inactive a site (C55/58S) and/or deletion of the C-terminal ER retention signal (ΔKDEL). ( c ) Immunoblot of PrP C , P4HB, and actin levels in cell lysates from CAD5 cells transiently transfected with empty vector (EV) or with WT or mutant P4HB for 48 h. ( d ) Quantification of PrP C and P4HB levels in lysates from transiently transfected CAD5 cells (n = 4 independent replicates). ( e ) Immunoblot of secreted PrP and P4HB levels in the conditioned medium from CAD5 cells transiently transfected with EV or with WT or mutant P4HB for 48 h. As a control, the membrane was stained for total protein following transfer using Ponceau S. ( f ) Representative immunoblots for proteinase K (PK)-resistant PrP Sc (PrP res ) levels in lysates from either RML or 22L prion-infected CAD5 cells transiently transfected with EV or with WT or mutant P4HB for 48 h. ( g ) Quantification of PrP res levels in transiently transfected RML or 22L prion-infected CAD5 cells (n = 4 independent replicates). In panels d and g, graphs display mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Incubation, Transfection, Plasmid Preparation, Mutagenesis, Control, Membrane, Staining, Infection

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Immunoblot of secreted P4HB levels in the conditioned medium (CM) from CAD5-PrP -/- cells transiently transfected with empty vector (EV) or with mutant P4HB variants lacking the C-terminal ER retention signal. As a control, the membrane was stained for total protein following transfer using Ponceau S. ( b ) Schematic of the experiment in which CM from transiently transfected CAD5-PrP -/- cells is applied to 22L prion-infected CAD5 cells for 72 h. ( c ) Representative immunoblot of proteinase K (PK)-resistant PrP Sc (PrP res ) levels in lysates from 22L prion-infected CAD5 cells treated with CM from CAD5-PrP -/- cells transiently transfected with EV or secreted P4HB variants. ( d ) Quantification of PrP res levels in lysates from 22L prion-infected CAD5 cells treated with CM from CAD5-PrP -/- cells transiently transfected with EV or secreted P4HB variants (n = 7 independent replicates). The graph displays mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Control, Membrane, Staining, Infection

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) Schematic of the experiment in which conditioned medium (CM) from transiently transfected CAD5-PrP -/- cells is applied to RML prion-infected CAD5 cells for 72 h. ( b ) Representative immunoblot of proteinase K (PK)-resistant PrP Sc (PrP res ) levels in lysates from RML prion-infected CAD5 cells treated with CM from CAD5-PrP -/- cells transiently transfected with empty vector (EV) or secreted P4HB variants. ( d ) Quantification of PrP res levels in lysates from RML prion-infected CAD5 cells treated with CM from CAD5-PrP -/- cells transiently transfected with EV or secreted P4HB variants (n = 7 independent replicates). The graph displays mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Transfection, Infection, Western Blot, Plasmid Preparation

Journal: bioRxiv

Article Title: The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrP C Levels and Prion Replication

doi: 10.64898/2025.12.01.691611

Figure Lengend Snippet: ( a ) P4HB helps to maintain the disulfide bond in PrP C . When P4HB is depleted or its disulfide isomerase activity is inhibited, the disulfide bond in PrP C forms less efficiently causing PrP C to become misfolded and degraded, which leads to lower steady-state PrP C levels. ( b ) P4HB functions as a chaperone that potentiates prion replication. In prion-infected cells, P4HB may act as a scaffold that facilitates interactions between PrP C and PrP Sc . ( c ) When P4HB levels are decreased, both the isomerase and chaperone activities of P4HB are decreased, leading to lower PrP C levels and less prion replication. When the catalytic a domain is inhibited, the chaperone activity of P4HB increases, leading to increased prion replication despite lower PrP C levels.

Article Snippet: To prepare the P4HB expression plasmids, WT mouse P4HB cDNA (#MG50638-UT, SinoBiological) or the ΔKDEL mutant was inserted between the BamHI and NotI sites of pcDNA3.

Techniques: Activity Assay, Infection