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rabbit anti xbp1s antibody (Proteintech)

Name: rabbit anti xbp1s antibody
Brand: Proteintech
Rating: 95 (91 reviews)

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Proteintech rabbit anti xbp1s antibody

The danger sensing UPR activates NLRP3 inflammasome components and the EBV lytic cycle in DLBCL. ( A and B ) VAL cells were pre-incubated with different concentrations of tunicamycin (TM) or 4μ8C for 2 h and then left untreated or treated with NaB for another 24 h. Cells were analyzed for ZEBRA expression by flow cytometry ( A ). Aggregate data from biological triplicates of the experiment in A are presented in B. ( C ) VAL cells were exposed to NaB + TPA for different lengths of time and collected for immunoblotting. ( D , E ) VAL cells were transfected with control siRNA (siCtrl) or siRNA targeting XBP1 (si XBP1 #1, #2). After 20 h, cells were collected for RT-qPCR analysis of XBP1 , NLRP3 and TXNIP transcripts ( D ), or induced with NaB for another 24 h for immunoblotting ( E ). ( F – H ) VAL cells were transfected with siCtrl + empty vector (EV), siCtrl + <t>XBP1S</t> plasmid, si XBP1 + EV or si XBP1 + XBP1S plasmid. Twenty-four hours later, cells were collected for RT-qPCR analysis of XBP1 and BZLF1 transcripts ( F ), or induced with NaB for another 24 h and collected for flow cytometry analysis ( G ). ( H ) Aggregate data of ZEBRA + cells from biological triplicates of the experiment in panel (G) are presented in panel (H). Error bars, SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Rabbit Anti Xbp1s Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 91 article reviews

rabbit anti xbp1s antibody - by Bioz Stars, 2026-02

95/100 stars

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1) Product Images from "Leveraging the interconnected unfolded protein response and NLRP3 inflammasome pathways to reactivate Epstein–Barr virus in diffuse large B-cell lymphomas"

Article Title: Leveraging the interconnected unfolded protein response and NLRP3 inflammasome pathways to reactivate Epstein–Barr virus in diffuse large B-cell lymphomas

Journal: NAR Cancer

doi: 10.1093/narcan/zcaf017

Figure Legend Snippet: The danger sensing UPR activates NLRP3 inflammasome components and the EBV lytic cycle in DLBCL. ( A and B ) VAL cells were pre-incubated with different concentrations of tunicamycin (TM) or 4μ8C for 2 h and then left untreated or treated with NaB for another 24 h. Cells were analyzed for ZEBRA expression by flow cytometry ( A ). Aggregate data from biological triplicates of the experiment in A are presented in B. ( C ) VAL cells were exposed to NaB + TPA for different lengths of time and collected for immunoblotting. ( D , E ) VAL cells were transfected with control siRNA (siCtrl) or siRNA targeting XBP1 (si XBP1 #1, #2). After 20 h, cells were collected for RT-qPCR analysis of XBP1 , NLRP3 and TXNIP transcripts ( D ), or induced with NaB for another 24 h for immunoblotting ( E ). ( F – H ) VAL cells were transfected with siCtrl + empty vector (EV), siCtrl + XBP1S plasmid, si XBP1 + EV or si XBP1 + XBP1S plasmid. Twenty-four hours later, cells were collected for RT-qPCR analysis of XBP1 and BZLF1 transcripts ( F ), or induced with NaB for another 24 h and collected for flow cytometry analysis ( G ). ( H ) Aggregate data of ZEBRA + cells from biological triplicates of the experiment in panel (G) are presented in panel (H). Error bars, SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Techniques Used: Incubation, Expressing, Flow Cytometry, Western Blot, Transfection, Control, Quantitative RT-PCR, Plasmid Preparation

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Journal: NAR Cancer

Article Title: Leveraging the interconnected unfolded protein response and NLRP3 inflammasome pathways to reactivate Epstein–Barr virus in diffuse large B-cell lymphomas

doi: 10.1093/narcan/zcaf017

Figure Lengend Snippet: The danger sensing UPR activates NLRP3 inflammasome components and the EBV lytic cycle in DLBCL. ( A and B ) VAL cells were pre-incubated with different concentrations of tunicamycin (TM) or 4μ8C for 2 h and then left untreated or treated with NaB for another 24 h. Cells were analyzed for ZEBRA expression by flow cytometry ( A ). Aggregate data from biological triplicates of the experiment in A are presented in B. ( C ) VAL cells were exposed to NaB + TPA for different lengths of time and collected for immunoblotting. ( D , E ) VAL cells were transfected with control siRNA (siCtrl) or siRNA targeting XBP1 (si XBP1 #1, #2). After 20 h, cells were collected for RT-qPCR analysis of XBP1 , NLRP3 and TXNIP transcripts ( D ), or induced with NaB for another 24 h for immunoblotting ( E ). ( F – H ) VAL cells were transfected with siCtrl + empty vector (EV), siCtrl + XBP1S plasmid, si XBP1 + EV or si XBP1 + XBP1S plasmid. Twenty-four hours later, cells were collected for RT-qPCR analysis of XBP1 and BZLF1 transcripts ( F ), or induced with NaB for another 24 h and collected for flow cytometry analysis ( G ). ( H ) Aggregate data of ZEBRA + cells from biological triplicates of the experiment in panel (G) are presented in panel (H). Error bars, SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The following antibodies were used: mouse anti-ZEBRA BZ1 antibody (generously provided by Paul Farrell, Imperial College London), mouse anti-β-actin antibody (AC-15, Sigma–Aldrich), rabbit anti-NLRP3 antibody (19771-1, Proteintech), rabbit anti-TXNIP antibody (14715, Cell Signaling Technology), rabbit anti-Caspase 1 (Cleaved Asp210) antibody ( PA538099 , Thermo Fisher Scientific), rabbit anti-Caspase 1 antibody ( PA587536 , Thermo Fisher Scientific), rabbit anti-XBP1S antibody (24868, Proteintech), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (626520, Thermo Fisher Scientific), and HRP-conjugated goat anti-rabbit IgG (31460, Thermo Fisher Scientific).

Techniques: Incubation, Expressing, Flow Cytometry, Western Blot, Transfection, Control, Quantitative RT-PCR, Plasmid Preparation

(A) Schematic of the SPLiCR-seq platform. The SPLiCR-seq vector enables the co-transcription of a splicing reporter and sgRNA driven by the EF1α promoter, while sgRNA expression for gene perturbation is driven by the U6 promoter. Paired-end sequencing links the splicing phenotype of the reporter to the corresponding sgRNA identity. (B) Diagram of the IRE1/XBP1 pathway. Under ER stress, IRE1’s RNase domain cleaves unspliced XBP1 mRNA ( XBP1u ), removing a 26-nucleotide intron. The resulting spliced XBP1 mRNA ( XBP1s ) is translated into a potent transcription factor that induces the expression of ER quality control genes to mitigate stress. (C) Design of the SPLiCR-seq vector for XBP1 splicing. The reporter contains the 26-nucleotide intron and 106-nucleotide 5’ and 32-nucleotide 3’ flanking sequences from XBP1u . (D, E) Dose-dependent splicing of endogenous XBP1 and the XBP1 reporter in the SPLiCR-seq vector in HEK293T cells treated with increasing concentrations of either Tg for 3 hours ( D ) or Tm for 6 hours ( E ), as assessed by RT-PCR. Spliced and unspliced products are indicated. (I) (F) Time-course analysis of XBP1 splicing dynamics for endogenous XBP1 and the XBP1 reporter in the SPLiCR-seq vector in HEK293T cells treated with Tg (100 nM or 500 nM) for the indicated durations, as assessed by RT-PCR. Spliced and unspliced products are indicated. (G, H) Quantification of time-course splicing levels for endogenous XBP1 ( G ) and XBP1 reporter ( H ) using grayscale analysis of RT-PCR bands. (I) Representative RT-PCR analysis showing splicing of endogenous XBP1 and XBP1 reporter in SPLiCR-seq vectors expressing a control, ERN1 or EIF2AK3 sgRNA in CRISPRi-HEK293T cells. (J, K) Quantification of endogenous XBP1 splicing ( J ) and XBP1 reporter splicing ( K ) from ( I ) (mean ± SD, n = 2 biological replicates).

Journal: bioRxiv

Article Title: SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1-XBP1 Signaling Under ER Stress

doi: 10.1101/2025.05.20.655206

Figure Lengend Snippet: (A) Schematic of the SPLiCR-seq platform. The SPLiCR-seq vector enables the co-transcription of a splicing reporter and sgRNA driven by the EF1α promoter, while sgRNA expression for gene perturbation is driven by the U6 promoter. Paired-end sequencing links the splicing phenotype of the reporter to the corresponding sgRNA identity. (B) Diagram of the IRE1/XBP1 pathway. Under ER stress, IRE1’s RNase domain cleaves unspliced XBP1 mRNA ( XBP1u ), removing a 26-nucleotide intron. The resulting spliced XBP1 mRNA ( XBP1s ) is translated into a potent transcription factor that induces the expression of ER quality control genes to mitigate stress. (C) Design of the SPLiCR-seq vector for XBP1 splicing. The reporter contains the 26-nucleotide intron and 106-nucleotide 5’ and 32-nucleotide 3’ flanking sequences from XBP1u . (D, E) Dose-dependent splicing of endogenous XBP1 and the XBP1 reporter in the SPLiCR-seq vector in HEK293T cells treated with increasing concentrations of either Tg for 3 hours ( D ) or Tm for 6 hours ( E ), as assessed by RT-PCR. Spliced and unspliced products are indicated. (I) (F) Time-course analysis of XBP1 splicing dynamics for endogenous XBP1 and the XBP1 reporter in the SPLiCR-seq vector in HEK293T cells treated with Tg (100 nM or 500 nM) for the indicated durations, as assessed by RT-PCR. Spliced and unspliced products are indicated. (G, H) Quantification of time-course splicing levels for endogenous XBP1 ( G ) and XBP1 reporter ( H ) using grayscale analysis of RT-PCR bands. (I) Representative RT-PCR analysis showing splicing of endogenous XBP1 and XBP1 reporter in SPLiCR-seq vectors expressing a control, ERN1 or EIF2AK3 sgRNA in CRISPRi-HEK293T cells. (J, K) Quantification of endogenous XBP1 splicing ( J ) and XBP1 reporter splicing ( K ) from ( I ) (mean ± SD, n = 2 biological replicates).

Article Snippet: The antibodies used in this study are summarized here: anti-PERK (Proteintech, 20582-1-AP), anti-Phospho-IRE1 (Abcam, ab124945), anti-IRE1 (CST, 3294), anti-GADD34 (Proteintech, 10449-1-AP), anti-XBP1 (CST, 12782), anti-ATF4 (Proteintech, 28657-1-AP), anti-Phospho-eIF2α (CST, 9721S), anti-eIF2α (CST, 5324), anti-GAPDH (Proteintech, HRP-60004), anti-Flag (Absin, Abs830014), anti-HA (Sigma, H3663), anti-GFP (Ray Antibody, RM1108).

Techniques: Plasmid Preparation, Expressing, Sequencing, Control, Reverse Transcription Polymerase Chain Reaction

(A) Schematic illustration of the SPLiCR screen workflow. HEK293T cells and iPSCs expressing the CRISPRi machinery were transduced with an sgRNA library targeting 1,350 human RNA-binding proteins (MOI < 0.3). After puromycin selection and cell expansion, cells were treated with Tg for 3 hours to induce ER stress. RNA was extracted, reverse-transcribed, and regions containing the XBP1 splicing reporter and sgRNA were PCR-amplified to generate paired-end NGS libraries. For each sgRNA, splicing phenotypes were calculated as the ratio of spliced reads (Si) to unspliced reads (Ui), and normalized to the median control sgRNA levels. The screen was performed in two biological replicates. (B, C) Volcano plots showing sgRNA phenotypes from the SPLiCR-seq screens in HEK293T cells and iPSC ( C ). sgRNAs targeting ERN1 are highlighted in dark blue. (I) (D) Comparison of gene knockdown phenotypes from HEK293T and iPSC SPLiCR-seq screens, revealing both common and cell-type-specific regulators of XBP1 splicing. (E, F) Distributions of phenotype scores for non-targeting control sgRNAs (gray) and sgRNAs targeting selected negative (blue) and positive (red) hits in HEK293T ( E ) and iPSC ( F ) SPLiCR-seq screens. (G) Representative RT-PCR analysis showing XBP1 splicing in HEK293T cells following CALR or UPF3B knockdown with two independent sgRNAs. The ratios of spliced to unspliced XBP1 ( XBP1s / XBP1u ) are indicated at the bottom. (H) Quantification of XBP1 splicing levels for CALR and UPF3B knockdown in HEK293T cells, normalized to control sgRNA (mean ± SD, n = 4 technical replicates). (I) Western blot showing protein levels of XBP1s, IRE1 and phosphorylated IRE1 (p-IRE1) in HEK293T cells under ER stress conditions following knockdown of CALR or UPF3B . GAPDH was used as a loading control. (J) Co-immunoprecipitation showing a direct physical interaction between CALR and IRE1. (K-N) Representative RT-PCR analysis showing XBP1 splicing under ER stress following DBR1 knockdown in HEK293T cells ( K ) and iPSCs ( M ), with splicing levels indicated at the bottom. Quantification of RT-PCR results showing the effect of DBR1 knockdown on XBP1 splicing in HEK293T cells ( L ) and iPSCs ( N ). Splicing levels are normalized to control sgRNA levels (mean ± SD, n = 3 technical replicates). Student’s t-test: **p < 0.01; ns, not significant.

Journal: bioRxiv

Article Title: SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1-XBP1 Signaling Under ER Stress

doi: 10.1101/2025.05.20.655206

Figure Lengend Snippet: (A) Schematic illustration of the SPLiCR screen workflow. HEK293T cells and iPSCs expressing the CRISPRi machinery were transduced with an sgRNA library targeting 1,350 human RNA-binding proteins (MOI < 0.3). After puromycin selection and cell expansion, cells were treated with Tg for 3 hours to induce ER stress. RNA was extracted, reverse-transcribed, and regions containing the XBP1 splicing reporter and sgRNA were PCR-amplified to generate paired-end NGS libraries. For each sgRNA, splicing phenotypes were calculated as the ratio of spliced reads (Si) to unspliced reads (Ui), and normalized to the median control sgRNA levels. The screen was performed in two biological replicates. (B, C) Volcano plots showing sgRNA phenotypes from the SPLiCR-seq screens in HEK293T cells and iPSC ( C ). sgRNAs targeting ERN1 are highlighted in dark blue. (I) (D) Comparison of gene knockdown phenotypes from HEK293T and iPSC SPLiCR-seq screens, revealing both common and cell-type-specific regulators of XBP1 splicing. (E, F) Distributions of phenotype scores for non-targeting control sgRNAs (gray) and sgRNAs targeting selected negative (blue) and positive (red) hits in HEK293T ( E ) and iPSC ( F ) SPLiCR-seq screens. (G) Representative RT-PCR analysis showing XBP1 splicing in HEK293T cells following CALR or UPF3B knockdown with two independent sgRNAs. The ratios of spliced to unspliced XBP1 ( XBP1s / XBP1u ) are indicated at the bottom. (H) Quantification of XBP1 splicing levels for CALR and UPF3B knockdown in HEK293T cells, normalized to control sgRNA (mean ± SD, n = 4 technical replicates). (I) Western blot showing protein levels of XBP1s, IRE1 and phosphorylated IRE1 (p-IRE1) in HEK293T cells under ER stress conditions following knockdown of CALR or UPF3B . GAPDH was used as a loading control. (J) Co-immunoprecipitation showing a direct physical interaction between CALR and IRE1. (K-N) Representative RT-PCR analysis showing XBP1 splicing under ER stress following DBR1 knockdown in HEK293T cells ( K ) and iPSCs ( M ), with splicing levels indicated at the bottom. Quantification of RT-PCR results showing the effect of DBR1 knockdown on XBP1 splicing in HEK293T cells ( L ) and iPSCs ( N ). Splicing levels are normalized to control sgRNA levels (mean ± SD, n = 3 technical replicates). Student’s t-test: **p < 0.01; ns, not significant.

Techniques: Expressing, Transduction, RNA Binding Assay, Selection, Reverse Transcription, Amplification, Control, Comparison, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation

(A) Schematic of the genome-wide SPLiCR-seq screen workflow. CRISPRi-HEK293T cells were transduced with a genome-wide sgRNA library targeting 11,120 genes that are expressed in HEK293T cells (MOI < 0.3). Following puromycin selection and cell expansion, cells were treated with either Tg or Tm for 24 hours to induce ER stress. RNA was extracted and SPLiCR libraries were prepared for NGS. (B, C) Volcano plots showing gene knockdown phenotypes from genome-wide screens in HEK293T cells treated with Tg ( B ) or Tm ( C ). Key known regulators of the IRE1-XBP1 pathway are labeled, with PPP1R15A , a novel regulator investigated in this study, highlighted in bold. (D) Comparison of gene knockdown phenotypes between Tg and Tm screens, showing a strong overlap in common regulators of XBP1 splicing as well as stressor-specific hits. (E, F) Functional categorization of hits from the genome-wide screens under Tg ( E ) and Tm ( F ) treatment. Red circles, positive hits; blue circles, negative hits. (G) GO Biological Process enrichment analysis of the top 100 positive hits (red) and top 100 negative hits (blue) for Tg (left) and Tm (right) treatments.

Journal: bioRxiv

Article Title: SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1-XBP1 Signaling Under ER Stress

doi: 10.1101/2025.05.20.655206

Figure Lengend Snippet: (A) Schematic of the genome-wide SPLiCR-seq screen workflow. CRISPRi-HEK293T cells were transduced with a genome-wide sgRNA library targeting 11,120 genes that are expressed in HEK293T cells (MOI < 0.3). Following puromycin selection and cell expansion, cells were treated with either Tg or Tm for 24 hours to induce ER stress. RNA was extracted and SPLiCR libraries were prepared for NGS. (B, C) Volcano plots showing gene knockdown phenotypes from genome-wide screens in HEK293T cells treated with Tg ( B ) or Tm ( C ). Key known regulators of the IRE1-XBP1 pathway are labeled, with PPP1R15A , a novel regulator investigated in this study, highlighted in bold. (D) Comparison of gene knockdown phenotypes between Tg and Tm screens, showing a strong overlap in common regulators of XBP1 splicing as well as stressor-specific hits. (E, F) Functional categorization of hits from the genome-wide screens under Tg ( E ) and Tm ( F ) treatment. Red circles, positive hits; blue circles, negative hits. (G) GO Biological Process enrichment analysis of the top 100 positive hits (red) and top 100 negative hits (blue) for Tg (left) and Tm (right) treatments.

Techniques: Genome Wide, Transduction, Selection, Knockdown, Labeling, Comparison, Functional Assay

(A) Schematic of the batch validation SPLiCR-seq screen workflow. (B) Heatmap showing gene knockdown phenotypes from the validation screens (top), compared to the primary screens (bottom). Genes are hierarchical clustered based on their validation screen phenotypes. Genes selected for individual validation are highlighted in blue (negative hits) and red (positive hits). (C-F) RT-PCR validations of XBP1 splicing in HEK293T cells under Tg- or Tm-induced ER stress following knockdown of SLC35B1 ( C ), MAN2A2 ( D ), SEC24A ( E ), PREB ( E ) and PPP1R15A ( F ). Splicing ratios are indicated below each lane. (G) Time-course RT-PCR analysis showing XBP1 splicing in PPP1R15A knockdown cells treated with Tg (500 nM) for the indicated durations. Splicing ratios are indicated below each lane. (H) RT-PCR analysis of XBP1 splicing in control and PPP1R15A knockdown HEK293T cells expressing either empty vector or GADD34 vector. Splicing ratios are indicated below each lane. (I) Western blot analysis of p-IRE1, total IRE1, and XBP1s protein levels in control and PPP1R15A knockdown cells. GAPDH was used as a loading control. (J) Western blot analysis of XBP1s protein levels in control and PPP1R15A knockout cells. GAPDH was used as a loading control. (K, L) RT-PCR analysis of XBP1 splicing in HEK293T cells treated with varying concentrations of Sephin1 ( K ) and at varying durations of Tm treatment ( L ). Splicing ratios are indicated below each lane.

Journal: bioRxiv

Article Title: SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1-XBP1 Signaling Under ER Stress

doi: 10.1101/2025.05.20.655206

Figure Lengend Snippet: (A) Schematic of the batch validation SPLiCR-seq screen workflow. (B) Heatmap showing gene knockdown phenotypes from the validation screens (top), compared to the primary screens (bottom). Genes are hierarchical clustered based on their validation screen phenotypes. Genes selected for individual validation are highlighted in blue (negative hits) and red (positive hits). (C-F) RT-PCR validations of XBP1 splicing in HEK293T cells under Tg- or Tm-induced ER stress following knockdown of SLC35B1 ( C ), MAN2A2 ( D ), SEC24A ( E ), PREB ( E ) and PPP1R15A ( F ). Splicing ratios are indicated below each lane. (G) Time-course RT-PCR analysis showing XBP1 splicing in PPP1R15A knockdown cells treated with Tg (500 nM) for the indicated durations. Splicing ratios are indicated below each lane. (H) RT-PCR analysis of XBP1 splicing in control and PPP1R15A knockdown HEK293T cells expressing either empty vector or GADD34 vector. Splicing ratios are indicated below each lane. (I) Western blot analysis of p-IRE1, total IRE1, and XBP1s protein levels in control and PPP1R15A knockdown cells. GAPDH was used as a loading control. (J) Western blot analysis of XBP1s protein levels in control and PPP1R15A knockout cells. GAPDH was used as a loading control. (K, L) RT-PCR analysis of XBP1 splicing in HEK293T cells treated with varying concentrations of Sephin1 ( K ) and at varying durations of Tm treatment ( L ). Splicing ratios are indicated below each lane.

Techniques: Biomarker Discovery, Knockdown, Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Plasmid Preparation, Western Blot, Knock-Out

(A) Western blot analysis of PERK, phosphorylated eIF2α (p-eIF2α) and total eIF2α levels in control and PPP1R15A knockdown HEK293T cells treated with Tg (500 nM) for the indicated durations (0, 6, or 24 hours). GAPDH was used as a loading control. (B) Western blot analysis of PERK, p-eIF2α, total eIF2α, p-IRE1, total IRE1, XBP1s and ATF4 in HEK293T cells treated with increasing concentrations of Sephin1 (0, 1, 5, or 10 μM) in the presence or absence of Tm (2 μM, 24 hours). GAPDH was used as a loading control. (C) Western blot analysis of p-eIF2α levels in cells with GADD34 overexpression via transient transfection (left) or lentiviral infection (right). GAPDH was used as a loading control. (D, E) RT-PCR analysis of XBP1 splicing in control and EIF2S1 (eIF2α) knockdown cells ( D ), and EIF2S1 partial knockout cells ( E ) treated with Tm (2 μM, 24 h) and Sephin1 (5 μM, 24 h) as indicated. Splicing ratios are indicated below each lane. (F) Western blot analysis of p-eIF2α, total eIF2α, p-IRE1, total IRE1 and XBP1s in control and EIF2S1 partial knockout HEK293T cells treated with Tm (2 μM, 24 h) and Sephin1 (5 μM, 24 h) as indicated. GAPDH was used as a loading control. (G) RT-PCR analysis of XBP1 splicing in HEK293T cells treated with Tm (2 μM, 24 h), Sephin1 (5 μM, 24 h), and ISRIB (1 μM, added 3 h before collection) as indicated. Splicing ratios are indicated below each lane.

Journal: bioRxiv

Article Title: SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1-XBP1 Signaling Under ER Stress

doi: 10.1101/2025.05.20.655206

Figure Lengend Snippet: (A) Western blot analysis of PERK, phosphorylated eIF2α (p-eIF2α) and total eIF2α levels in control and PPP1R15A knockdown HEK293T cells treated with Tg (500 nM) for the indicated durations (0, 6, or 24 hours). GAPDH was used as a loading control. (B) Western blot analysis of PERK, p-eIF2α, total eIF2α, p-IRE1, total IRE1, XBP1s and ATF4 in HEK293T cells treated with increasing concentrations of Sephin1 (0, 1, 5, or 10 μM) in the presence or absence of Tm (2 μM, 24 hours). GAPDH was used as a loading control. (C) Western blot analysis of p-eIF2α levels in cells with GADD34 overexpression via transient transfection (left) or lentiviral infection (right). GAPDH was used as a loading control. (D, E) RT-PCR analysis of XBP1 splicing in control and EIF2S1 (eIF2α) knockdown cells ( D ), and EIF2S1 partial knockout cells ( E ) treated with Tm (2 μM, 24 h) and Sephin1 (5 μM, 24 h) as indicated. Splicing ratios are indicated below each lane. (F) Western blot analysis of p-eIF2α, total eIF2α, p-IRE1, total IRE1 and XBP1s in control and EIF2S1 partial knockout HEK293T cells treated with Tm (2 μM, 24 h) and Sephin1 (5 μM, 24 h) as indicated. GAPDH was used as a loading control. (G) RT-PCR analysis of XBP1 splicing in HEK293T cells treated with Tm (2 μM, 24 h), Sephin1 (5 μM, 24 h), and ISRIB (1 μM, added 3 h before collection) as indicated. Splicing ratios are indicated below each lane.

Techniques: Western Blot, Control, Knockdown, Over Expression, Transfection, Infection, Reverse Transcription Polymerase Chain Reaction, Knock-Out

(A) Schematic diagram of the ex-vivo CAR-T cell exhaustion model. (B) Representative agarose gel showing RT-PCR products of endogenous XBP1 splicing in CAR-T cells repetitively co-cultured with tumor cells for 3, 6 and 9 days in the presence of DMSO or Sephin1. (C) XBP1 splicing level in CAR-T cells repetitively co-cultured with tumor cells for 3, 6 and 9 days in the presence of DMSO or Sephin1 and normalized to that of the DMSO treatment in day 0 and presented as fold change. Data are presented as mean ± SD, paired Student’s t-test; ***P<0.0001, n = 4 technical replicates). (D, E) Intracellular cytokine staining of IFN-γ and TNF-α in CAR-T cells repetitively co-cultured with tumor cells for 3, 6 and 9 days in the presence of DMSO or Sephin1 and the percentage of IFN-γ + /TNF-α + and IFN-γ - /TNF-α - in CAR-T cells at day 9. (Data are presented as mean ± SEM, paired Student’s t-test; *P < 0.05, n = 5 experiments). (F, G) Expression of of IFN-γ and TNF-α in CAR-T cells at day 9 and the mean fluorescent intensity (MFI) of IFN-γ and TNF-α in cells stained positive for respective cytokine. (Data are presented as mean ± SEM, paired Student’s t-test; *P < 0.05, n = 5 experiments). (H) Cell counts of CAR-T cells and BXPC3 cells at day 3, 6, 9 upon treatment of DMSO or Sephin1 (Data are presented as mean ± SEM, two-way ANOVA with Sidak multiple comparisons test; ns, not significant, *P < 0.05, **P < 0.01, n = 5 experiments).

Journal: bioRxiv

Article Title: SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1-XBP1 Signaling Under ER Stress

doi: 10.1101/2025.05.20.655206

Figure Lengend Snippet: (A) Schematic diagram of the ex-vivo CAR-T cell exhaustion model. (B) Representative agarose gel showing RT-PCR products of endogenous XBP1 splicing in CAR-T cells repetitively co-cultured with tumor cells for 3, 6 and 9 days in the presence of DMSO or Sephin1. (C) XBP1 splicing level in CAR-T cells repetitively co-cultured with tumor cells for 3, 6 and 9 days in the presence of DMSO or Sephin1 and normalized to that of the DMSO treatment in day 0 and presented as fold change. Data are presented as mean ± SD, paired Student’s t-test; ***P<0.0001, n = 4 technical replicates). (D, E) Intracellular cytokine staining of IFN-γ and TNF-α in CAR-T cells repetitively co-cultured with tumor cells for 3, 6 and 9 days in the presence of DMSO or Sephin1 and the percentage of IFN-γ + /TNF-α + and IFN-γ - /TNF-α - in CAR-T cells at day 9. (Data are presented as mean ± SEM, paired Student’s t-test; *P < 0.05, n = 5 experiments). (F, G) Expression of of IFN-γ and TNF-α in CAR-T cells at day 9 and the mean fluorescent intensity (MFI) of IFN-γ and TNF-α in cells stained positive for respective cytokine. (Data are presented as mean ± SEM, paired Student’s t-test; *P < 0.05, n = 5 experiments). (H) Cell counts of CAR-T cells and BXPC3 cells at day 3, 6, 9 upon treatment of DMSO or Sephin1 (Data are presented as mean ± SEM, two-way ANOVA with Sidak multiple comparisons test; ns, not significant, *P < 0.05, **P < 0.01, n = 5 experiments).

Techniques: Ex Vivo, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Staining, Expressing

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