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scotin knockout cell generation  (Santa Cruz Biotechnology)


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    Santa Cruz Biotechnology scotin knockout cell generation
    Scotin Knockout Cell Generation, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Figure 1. <t>SCOTIN</t> overexpression induces cell death and activates the ER stress response (A) PARP1 cleavage in HeLa cells expressing SCOTIN-MYC was evaluated by western blotting at the indicated times after transfection. See also Figure S1B. (B) Cleavage of PARP1, caspase-3, and caspase-8 following SCOTIN-MYC overexpression was evaluated by western blotting 24 h after transfection. (C) The mRNA levels of XBP1s, CHOP, and GRP78 in HeLa cells expressing SCOTIN-MYC were measured by RT-qPCR. The protein levels of SCOTIN-MYC were measured by western blotting. The error bars indicate the means ± SDs from triplicate experiments. (D) The protein levels of XBP1s and CHOP in HeLa cells expressing SCOTIN-MYC were measured by western blotting. (E–G) HeLa cells were transfected with the indicated constructs and treated with tunicamycin (1 mg/mL) for the indicated times before cell harvesting. Protein levels were measured by western blotting. (H) IRE1-3F6HGFP focus formation was induced by tunicamycin (1 mg/mL) treatment for 4 h in HeLa cells. Scale bars, 5 mm. (I) Statistical analysis of IRE1-3F6HGFP focus formation in SCOTIN-MYC-expressing HeLa cells. The graph presents the means ± SDs of three independent experiments.
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    Figure 1. <t>SCOTIN</t> inhibits transport from ER to Golgi (A) Schematic illustration of proximal profiling of SCOTIN with BioID. (B) Gene ontology (GO) analysis of proteins identified in the proximal profiling. (C) Schematic illustration of the RUSH reporter system. (D) Cell surface trafficking of E-cadherin-SBP-EGFP was assessed after expression of indicated constructs followed by biotin treatment. (E) Live confocal images <t>of</t> <t>HeLa</t> cells transfected with the E-cadherin-SBP-EGFP with empty or SCOTIN-MYC plasmids during the first 20 min after biotin treatment. (F) (Top) Confocal images of HeLa cells expressing E-cadherin-SBP-EGFP with empty, or SCOTIN-MYC with or without biotin (15 min) followed by immuno- staining with MYC and giantin. (Bottom) The Mander’s coefficient of EGFP overlapping giantin for each cell (n = 6–12). (G) (Top) Confocal images of HeLa cells expressing SBP-EGFP-GPI with empty, or SCOTIN-MYC with or without biotin (7 min) followed by immunostaining with MYC and giantin. (Bottom) The Mander’s coefficient of EGFP overlapping GM130 for each cell (n = 15–17). (H) The trafficking of VSV-Gts045 from ER to Golgi was observed after temperature shift from 40C to 32C for 10 min. Confocal images of HeLa cells after im- munostaining of VSV-G and giantin (top), and Mander’s coefficient of VSV-G overlapping giantin (bottom) were shown (n = 6–8). All graphs are represented as mean ± SEM. All scale bars, 10mm. ***p < 0.001
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    Figure 1. SCOTIN overexpression induces cell death and activates the ER stress response (A) PARP1 cleavage in HeLa cells expressing SCOTIN-MYC was evaluated by western blotting at the indicated times after transfection. See also Figure S1B. (B) Cleavage of PARP1, caspase-3, and caspase-8 following SCOTIN-MYC overexpression was evaluated by western blotting 24 h after transfection. (C) The mRNA levels of XBP1s, CHOP, and GRP78 in HeLa cells expressing SCOTIN-MYC were measured by RT-qPCR. The protein levels of SCOTIN-MYC were measured by western blotting. The error bars indicate the means ± SDs from triplicate experiments. (D) The protein levels of XBP1s and CHOP in HeLa cells expressing SCOTIN-MYC were measured by western blotting. (E–G) HeLa cells were transfected with the indicated constructs and treated with tunicamycin (1 mg/mL) for the indicated times before cell harvesting. Protein levels were measured by western blotting. (H) IRE1-3F6HGFP focus formation was induced by tunicamycin (1 mg/mL) treatment for 4 h in HeLa cells. Scale bars, 5 mm. (I) Statistical analysis of IRE1-3F6HGFP focus formation in SCOTIN-MYC-expressing HeLa cells. The graph presents the means ± SDs of three independent experiments.

    Journal: Cell reports

    Article Title: Membrane-tethered SCOTIN condensates elicit an endoplasmic reticulum stress response by sequestering luminal BiP.

    doi: 10.1016/j.celrep.2025.115297

    Figure Lengend Snippet: Figure 1. SCOTIN overexpression induces cell death and activates the ER stress response (A) PARP1 cleavage in HeLa cells expressing SCOTIN-MYC was evaluated by western blotting at the indicated times after transfection. See also Figure S1B. (B) Cleavage of PARP1, caspase-3, and caspase-8 following SCOTIN-MYC overexpression was evaluated by western blotting 24 h after transfection. (C) The mRNA levels of XBP1s, CHOP, and GRP78 in HeLa cells expressing SCOTIN-MYC were measured by RT-qPCR. The protein levels of SCOTIN-MYC were measured by western blotting. The error bars indicate the means ± SDs from triplicate experiments. (D) The protein levels of XBP1s and CHOP in HeLa cells expressing SCOTIN-MYC were measured by western blotting. (E–G) HeLa cells were transfected with the indicated constructs and treated with tunicamycin (1 mg/mL) for the indicated times before cell harvesting. Protein levels were measured by western blotting. (H) IRE1-3F6HGFP focus formation was induced by tunicamycin (1 mg/mL) treatment for 4 h in HeLa cells. Scale bars, 5 mm. (I) Statistical analysis of IRE1-3F6HGFP focus formation in SCOTIN-MYC-expressing HeLa cells. The graph presents the means ± SDs of three independent experiments.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER A549 ATCC Cat# CCL-185 DLD-1 Korean Cell Line Bank Cat# 10221 HCT 116 Korean Cell Line Bank Cat# 10247 HeLa ATCC Cat# CCL-2 HeLa SCOTIN knockout (KO) This study N/A HepG2 ATCC Cat# HB-8065 HT-29 Korean Cell Line Bank Cat# 30038 Huh7 Korean Cell Line Bank Cat# 60104 SW480 Korean Cell Line Bank Cat# 10228 SW620 Korean Cell Line Bank Cat# 60068 U-2 OS Korean Cell Line Bank Cat# 30096 Recombinant DNA SCOTIN This study N/A SCOTIN-TC This study N/A SCOTIN-MYC This study N/A SCOTIN(D150-177)-MYC This study N/A SCOTIN(D29-43)-MYC This study N/A SCOTIN(D44-69)-MYC This study N/A SCOTIN(D70-105)-MYC This study N/A SCOTIN(D44-105)-MYC This study N/A SCOTIN(D29-43,D70-105)-MYC This study N/A SCOTIN(D29-69)-MYC This study N/A SCOTIN(D29-43,D150-177)-MYC This study N/A SCOTIN(D44-69,D150-177)-MYC This study N/A SCOTIN(D70-105,D150-177)-MYC This study N/A SCOTIN(D44-105,D150-177)-MYC This study N/A SCOTIN(D29-43,D70-105,D150-177)-MYC This study N/A SCOTIN(D29-69,D150-177)-MYC This study N/A SCOTIN(DCRD)-MYC This study N/A SCOTIN(DPRD)-MYC This study N/A SCOTIN(DPRD+(150–177))-MYC This study N/A SCOTIN(1–149)-MYC This study N/A SCOTIN(1–177)-MYC This study N/A SCOTIN(DPRD)-FUSN-MYC This study N/A SCOTIN(1–149)-FUSN-MYC This study N/A SCOTIN-APEX2 This study N/A mCherry-Cry2 This study N/A SCOTIN(CRD)-mCherry-Cry2 This study N/A 3xFLAG-ATF6 Chen et al.72 Addgene #11975 IRE1-3F6HGFP Peter Walter Lab (UCSF)29 N/A F-XBP1DDBD-venus Masayuki Miura (University of Tokyo)33 N/A BiP-EGFP-KDEL This study N/A BiP(K294F)-EGFP-KDEL This study N/A BiP(V461F)-EGFP-KDEL This study N/A BiP-(GGGGS)3-mNeonGreen-KDEL This study N/A SCOTIN gRNA/Cas9 knockout plasmid Santa Cruz Cat# sc-408226 SCOTIN HDR plasmid Santa Cruz Cat# sc-408226-HDR Cre Vector Santa Cruz Cat# sc-418923 (Continued on next page) 18 Cell Reports 44, 115297, February 25, 2025

    Techniques: Over Expression, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Construct, Cell Harvesting

    Figure 2. SCOTIN condensation is critical for inducing ER stress (A) The production of XBP1s and induction of CHOP in SCOTIN KO HeLa cells expressing SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC were evaluated by western blotting. (B) Representative confocal images of IRE1-3F6HGFP focus formation in SCOTIN KO HeLa cells expressing SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC. Scale bars, 5 mm. (C) Statistical analysis of IRE1-3F6HGFP focus formation in SCOTIN KO HeLa cells expressing SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC. The graph presents the means ± SDs of three independent experiments. (D) Images of representative flow cytometry density plots showing the production of F-XBP1DDBD-Venus in SCOTIN KO HeLa cells expressing SCOTIN(full)- MYC or SCOTIN(D150–177)-MYC. (E) Statistical analysis of the F-XBP1DDBD-Venus production data in (D). The error bars indicate the means ± SDs from triplicate experiments. The asterisks indicate the p values determined by an unpaired two-tailed t test. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001, ****p ˂ 0.0001.

    Journal: Cell reports

    Article Title: Membrane-tethered SCOTIN condensates elicit an endoplasmic reticulum stress response by sequestering luminal BiP.

    doi: 10.1016/j.celrep.2025.115297

    Figure Lengend Snippet: Figure 2. SCOTIN condensation is critical for inducing ER stress (A) The production of XBP1s and induction of CHOP in SCOTIN KO HeLa cells expressing SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC were evaluated by western blotting. (B) Representative confocal images of IRE1-3F6HGFP focus formation in SCOTIN KO HeLa cells expressing SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC. Scale bars, 5 mm. (C) Statistical analysis of IRE1-3F6HGFP focus formation in SCOTIN KO HeLa cells expressing SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC. The graph presents the means ± SDs of three independent experiments. (D) Images of representative flow cytometry density plots showing the production of F-XBP1DDBD-Venus in SCOTIN KO HeLa cells expressing SCOTIN(full)- MYC or SCOTIN(D150–177)-MYC. (E) Statistical analysis of the F-XBP1DDBD-Venus production data in (D). The error bars indicate the means ± SDs from triplicate experiments. The asterisks indicate the p values determined by an unpaired two-tailed t test. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001, ****p ˂ 0.0001.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER A549 ATCC Cat# CCL-185 DLD-1 Korean Cell Line Bank Cat# 10221 HCT 116 Korean Cell Line Bank Cat# 10247 HeLa ATCC Cat# CCL-2 HeLa SCOTIN knockout (KO) This study N/A HepG2 ATCC Cat# HB-8065 HT-29 Korean Cell Line Bank Cat# 30038 Huh7 Korean Cell Line Bank Cat# 60104 SW480 Korean Cell Line Bank Cat# 10228 SW620 Korean Cell Line Bank Cat# 60068 U-2 OS Korean Cell Line Bank Cat# 30096 Recombinant DNA SCOTIN This study N/A SCOTIN-TC This study N/A SCOTIN-MYC This study N/A SCOTIN(D150-177)-MYC This study N/A SCOTIN(D29-43)-MYC This study N/A SCOTIN(D44-69)-MYC This study N/A SCOTIN(D70-105)-MYC This study N/A SCOTIN(D44-105)-MYC This study N/A SCOTIN(D29-43,D70-105)-MYC This study N/A SCOTIN(D29-69)-MYC This study N/A SCOTIN(D29-43,D150-177)-MYC This study N/A SCOTIN(D44-69,D150-177)-MYC This study N/A SCOTIN(D70-105,D150-177)-MYC This study N/A SCOTIN(D44-105,D150-177)-MYC This study N/A SCOTIN(D29-43,D70-105,D150-177)-MYC This study N/A SCOTIN(D29-69,D150-177)-MYC This study N/A SCOTIN(DCRD)-MYC This study N/A SCOTIN(DPRD)-MYC This study N/A SCOTIN(DPRD+(150–177))-MYC This study N/A SCOTIN(1–149)-MYC This study N/A SCOTIN(1–177)-MYC This study N/A SCOTIN(DPRD)-FUSN-MYC This study N/A SCOTIN(1–149)-FUSN-MYC This study N/A SCOTIN-APEX2 This study N/A mCherry-Cry2 This study N/A SCOTIN(CRD)-mCherry-Cry2 This study N/A 3xFLAG-ATF6 Chen et al.72 Addgene #11975 IRE1-3F6HGFP Peter Walter Lab (UCSF)29 N/A F-XBP1DDBD-venus Masayuki Miura (University of Tokyo)33 N/A BiP-EGFP-KDEL This study N/A BiP(K294F)-EGFP-KDEL This study N/A BiP(V461F)-EGFP-KDEL This study N/A BiP-(GGGGS)3-mNeonGreen-KDEL This study N/A SCOTIN gRNA/Cas9 knockout plasmid Santa Cruz Cat# sc-408226 SCOTIN HDR plasmid Santa Cruz Cat# sc-408226-HDR Cre Vector Santa Cruz Cat# sc-418923 (Continued on next page) 18 Cell Reports 44, 115297, February 25, 2025

    Techniques: Expressing, Western Blot, Cytometry, Two Tailed Test

    Figure 3. Morphological signs of cellular stress in cells with SCOTIN condensates (A and B) HeLa cells were transfected with MYC-tagged, TC-tagged, or untagged SCOTIN. PARP1 cleavage (A) and production of XBP1s and induction of CHOP (B) were evaluated by western blotting. (C and D) CLEM images of HeLa cells expressing SCOTIN-TC. Scale bars, 5 mm. (E) Enlarged image of SCOTIN-TC puncta in the yellow box in (C). The 3D distribution of SCOTIN-TC was determined by reconstruction from 18 serial images. Scale bars, 1 mm. (F and G) Enlarged images of SCOTIN-TC puncta in the red box (ER and mitochondria) and green box (Golgi apparatus) in (C) and (D). Scale bars, 1 mm. (H–J) TEM images of HeLa cells expressing SCOTIN-APEX2 or the control vector. Scale bars, 1 mm. See also Figure S2.

    Journal: Cell reports

    Article Title: Membrane-tethered SCOTIN condensates elicit an endoplasmic reticulum stress response by sequestering luminal BiP.

    doi: 10.1016/j.celrep.2025.115297

    Figure Lengend Snippet: Figure 3. Morphological signs of cellular stress in cells with SCOTIN condensates (A and B) HeLa cells were transfected with MYC-tagged, TC-tagged, or untagged SCOTIN. PARP1 cleavage (A) and production of XBP1s and induction of CHOP (B) were evaluated by western blotting. (C and D) CLEM images of HeLa cells expressing SCOTIN-TC. Scale bars, 5 mm. (E) Enlarged image of SCOTIN-TC puncta in the yellow box in (C). The 3D distribution of SCOTIN-TC was determined by reconstruction from 18 serial images. Scale bars, 1 mm. (F and G) Enlarged images of SCOTIN-TC puncta in the red box (ER and mitochondria) and green box (Golgi apparatus) in (C) and (D). Scale bars, 1 mm. (H–J) TEM images of HeLa cells expressing SCOTIN-APEX2 or the control vector. Scale bars, 1 mm. See also Figure S2.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER A549 ATCC Cat# CCL-185 DLD-1 Korean Cell Line Bank Cat# 10221 HCT 116 Korean Cell Line Bank Cat# 10247 HeLa ATCC Cat# CCL-2 HeLa SCOTIN knockout (KO) This study N/A HepG2 ATCC Cat# HB-8065 HT-29 Korean Cell Line Bank Cat# 30038 Huh7 Korean Cell Line Bank Cat# 60104 SW480 Korean Cell Line Bank Cat# 10228 SW620 Korean Cell Line Bank Cat# 60068 U-2 OS Korean Cell Line Bank Cat# 30096 Recombinant DNA SCOTIN This study N/A SCOTIN-TC This study N/A SCOTIN-MYC This study N/A SCOTIN(D150-177)-MYC This study N/A SCOTIN(D29-43)-MYC This study N/A SCOTIN(D44-69)-MYC This study N/A SCOTIN(D70-105)-MYC This study N/A SCOTIN(D44-105)-MYC This study N/A SCOTIN(D29-43,D70-105)-MYC This study N/A SCOTIN(D29-69)-MYC This study N/A SCOTIN(D29-43,D150-177)-MYC This study N/A SCOTIN(D44-69,D150-177)-MYC This study N/A SCOTIN(D70-105,D150-177)-MYC This study N/A SCOTIN(D44-105,D150-177)-MYC This study N/A SCOTIN(D29-43,D70-105,D150-177)-MYC This study N/A SCOTIN(D29-69,D150-177)-MYC This study N/A SCOTIN(DCRD)-MYC This study N/A SCOTIN(DPRD)-MYC This study N/A SCOTIN(DPRD+(150–177))-MYC This study N/A SCOTIN(1–149)-MYC This study N/A SCOTIN(1–177)-MYC This study N/A SCOTIN(DPRD)-FUSN-MYC This study N/A SCOTIN(1–149)-FUSN-MYC This study N/A SCOTIN-APEX2 This study N/A mCherry-Cry2 This study N/A SCOTIN(CRD)-mCherry-Cry2 This study N/A 3xFLAG-ATF6 Chen et al.72 Addgene #11975 IRE1-3F6HGFP Peter Walter Lab (UCSF)29 N/A F-XBP1DDBD-venus Masayuki Miura (University of Tokyo)33 N/A BiP-EGFP-KDEL This study N/A BiP(K294F)-EGFP-KDEL This study N/A BiP(V461F)-EGFP-KDEL This study N/A BiP-(GGGGS)3-mNeonGreen-KDEL This study N/A SCOTIN gRNA/Cas9 knockout plasmid Santa Cruz Cat# sc-408226 SCOTIN HDR plasmid Santa Cruz Cat# sc-408226-HDR Cre Vector Santa Cruz Cat# sc-418923 (Continued on next page) 18 Cell Reports 44, 115297, February 25, 2025

    Techniques: Transfection, Western Blot, Expressing, Control, Plasmid Preparation

    Figure 4. BiP, an ER chaperone protein, is sequestered within immobile SCOTIN condensates (A) Confocal images of SCOTIN-MYC, IRE1-3F6HGFP, and BiP in HeLa cells. Scale bars, 5 mm. (B) Quantification data for colocalization of IRE1-3F6HGFP/BiP, IRE1-3F6HGFP/SCOTIN-MYC, and SCOTIN-MYC/BiP in (A) (n = 56). The error bars indicate the means ± SDs. See also Figure S3B. (C) Confocal images of SCOTIN-MYC, GRP94 and Sec61b in HeLa cells. Scale bars, 5 mm. (D) Confocal images of BiP in SCOTIN KO HeLa cells expressing SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC. Scale bars, 5 mm

    Journal: Cell reports

    Article Title: Membrane-tethered SCOTIN condensates elicit an endoplasmic reticulum stress response by sequestering luminal BiP.

    doi: 10.1016/j.celrep.2025.115297

    Figure Lengend Snippet: Figure 4. BiP, an ER chaperone protein, is sequestered within immobile SCOTIN condensates (A) Confocal images of SCOTIN-MYC, IRE1-3F6HGFP, and BiP in HeLa cells. Scale bars, 5 mm. (B) Quantification data for colocalization of IRE1-3F6HGFP/BiP, IRE1-3F6HGFP/SCOTIN-MYC, and SCOTIN-MYC/BiP in (A) (n = 56). The error bars indicate the means ± SDs. See also Figure S3B. (C) Confocal images of SCOTIN-MYC, GRP94 and Sec61b in HeLa cells. Scale bars, 5 mm. (D) Confocal images of BiP in SCOTIN KO HeLa cells expressing SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC. Scale bars, 5 mm

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER A549 ATCC Cat# CCL-185 DLD-1 Korean Cell Line Bank Cat# 10221 HCT 116 Korean Cell Line Bank Cat# 10247 HeLa ATCC Cat# CCL-2 HeLa SCOTIN knockout (KO) This study N/A HepG2 ATCC Cat# HB-8065 HT-29 Korean Cell Line Bank Cat# 30038 Huh7 Korean Cell Line Bank Cat# 60104 SW480 Korean Cell Line Bank Cat# 10228 SW620 Korean Cell Line Bank Cat# 60068 U-2 OS Korean Cell Line Bank Cat# 30096 Recombinant DNA SCOTIN This study N/A SCOTIN-TC This study N/A SCOTIN-MYC This study N/A SCOTIN(D150-177)-MYC This study N/A SCOTIN(D29-43)-MYC This study N/A SCOTIN(D44-69)-MYC This study N/A SCOTIN(D70-105)-MYC This study N/A SCOTIN(D44-105)-MYC This study N/A SCOTIN(D29-43,D70-105)-MYC This study N/A SCOTIN(D29-69)-MYC This study N/A SCOTIN(D29-43,D150-177)-MYC This study N/A SCOTIN(D44-69,D150-177)-MYC This study N/A SCOTIN(D70-105,D150-177)-MYC This study N/A SCOTIN(D44-105,D150-177)-MYC This study N/A SCOTIN(D29-43,D70-105,D150-177)-MYC This study N/A SCOTIN(D29-69,D150-177)-MYC This study N/A SCOTIN(DCRD)-MYC This study N/A SCOTIN(DPRD)-MYC This study N/A SCOTIN(DPRD+(150–177))-MYC This study N/A SCOTIN(1–149)-MYC This study N/A SCOTIN(1–177)-MYC This study N/A SCOTIN(DPRD)-FUSN-MYC This study N/A SCOTIN(1–149)-FUSN-MYC This study N/A SCOTIN-APEX2 This study N/A mCherry-Cry2 This study N/A SCOTIN(CRD)-mCherry-Cry2 This study N/A 3xFLAG-ATF6 Chen et al.72 Addgene #11975 IRE1-3F6HGFP Peter Walter Lab (UCSF)29 N/A F-XBP1DDBD-venus Masayuki Miura (University of Tokyo)33 N/A BiP-EGFP-KDEL This study N/A BiP(K294F)-EGFP-KDEL This study N/A BiP(V461F)-EGFP-KDEL This study N/A BiP-(GGGGS)3-mNeonGreen-KDEL This study N/A SCOTIN gRNA/Cas9 knockout plasmid Santa Cruz Cat# sc-408226 SCOTIN HDR plasmid Santa Cruz Cat# sc-408226-HDR Cre Vector Santa Cruz Cat# sc-418923 (Continued on next page) 18 Cell Reports 44, 115297, February 25, 2025

    Techniques: Expressing

    Figure 5. PRD-mediated condensation is needed for physical interaction between SCOTIN and the luminal BiP (A) Co-immunoprecipitation (coIP) of SCOTIN-MYC with endogenous BiP in HeLa cells. (B) CoIP of SCOTIN-MYC with BiP-EGFP-KDEL mutants in HeLa cells. (C) CoIP of SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC with endogenous BiP in SCOTIN KO HeLa cells. (D) Schematic of the CRD deletion mutant constructs of SCOTIN-MYC. (E–G) CoIP of SCOTIN-MYC mutants with endogenous BiP in SCOTIN KO HeLa cells.

    Journal: Cell reports

    Article Title: Membrane-tethered SCOTIN condensates elicit an endoplasmic reticulum stress response by sequestering luminal BiP.

    doi: 10.1016/j.celrep.2025.115297

    Figure Lengend Snippet: Figure 5. PRD-mediated condensation is needed for physical interaction between SCOTIN and the luminal BiP (A) Co-immunoprecipitation (coIP) of SCOTIN-MYC with endogenous BiP in HeLa cells. (B) CoIP of SCOTIN-MYC with BiP-EGFP-KDEL mutants in HeLa cells. (C) CoIP of SCOTIN(full)-MYC or SCOTIN(D150–177)-MYC with endogenous BiP in SCOTIN KO HeLa cells. (D) Schematic of the CRD deletion mutant constructs of SCOTIN-MYC. (E–G) CoIP of SCOTIN-MYC mutants with endogenous BiP in SCOTIN KO HeLa cells.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER A549 ATCC Cat# CCL-185 DLD-1 Korean Cell Line Bank Cat# 10221 HCT 116 Korean Cell Line Bank Cat# 10247 HeLa ATCC Cat# CCL-2 HeLa SCOTIN knockout (KO) This study N/A HepG2 ATCC Cat# HB-8065 HT-29 Korean Cell Line Bank Cat# 30038 Huh7 Korean Cell Line Bank Cat# 60104 SW480 Korean Cell Line Bank Cat# 10228 SW620 Korean Cell Line Bank Cat# 60068 U-2 OS Korean Cell Line Bank Cat# 30096 Recombinant DNA SCOTIN This study N/A SCOTIN-TC This study N/A SCOTIN-MYC This study N/A SCOTIN(D150-177)-MYC This study N/A SCOTIN(D29-43)-MYC This study N/A SCOTIN(D44-69)-MYC This study N/A SCOTIN(D70-105)-MYC This study N/A SCOTIN(D44-105)-MYC This study N/A SCOTIN(D29-43,D70-105)-MYC This study N/A SCOTIN(D29-69)-MYC This study N/A SCOTIN(D29-43,D150-177)-MYC This study N/A SCOTIN(D44-69,D150-177)-MYC This study N/A SCOTIN(D70-105,D150-177)-MYC This study N/A SCOTIN(D44-105,D150-177)-MYC This study N/A SCOTIN(D29-43,D70-105,D150-177)-MYC This study N/A SCOTIN(D29-69,D150-177)-MYC This study N/A SCOTIN(DCRD)-MYC This study N/A SCOTIN(DPRD)-MYC This study N/A SCOTIN(DPRD+(150–177))-MYC This study N/A SCOTIN(1–149)-MYC This study N/A SCOTIN(1–177)-MYC This study N/A SCOTIN(DPRD)-FUSN-MYC This study N/A SCOTIN(1–149)-FUSN-MYC This study N/A SCOTIN-APEX2 This study N/A mCherry-Cry2 This study N/A SCOTIN(CRD)-mCherry-Cry2 This study N/A 3xFLAG-ATF6 Chen et al.72 Addgene #11975 IRE1-3F6HGFP Peter Walter Lab (UCSF)29 N/A F-XBP1DDBD-venus Masayuki Miura (University of Tokyo)33 N/A BiP-EGFP-KDEL This study N/A BiP(K294F)-EGFP-KDEL This study N/A BiP(V461F)-EGFP-KDEL This study N/A BiP-(GGGGS)3-mNeonGreen-KDEL This study N/A SCOTIN gRNA/Cas9 knockout plasmid Santa Cruz Cat# sc-408226 SCOTIN HDR plasmid Santa Cruz Cat# sc-408226-HDR Cre Vector Santa Cruz Cat# sc-418923 (Continued on next page) 18 Cell Reports 44, 115297, February 25, 2025

    Techniques: Immunoprecipitation, Mutagenesis, Construct

    Figure 6. PRD-mediated condensation and BiP sequestration are functionally linked to the induction of ER stress responses (A) Confocal images of SCOTIN-MYC mutants and endogenous BiP in SCOTIN KO HeLa cells. Scale bars, 5 mm. (B) Quantification data for colocalization of BiP/SCOTIN-MYC in (A) (n = 30–119). The error bars indicate the means ± SDs. See also Figure S3D. (C) Confocal images of BiP-EGFP-KDEL mutants and SCOTIN-MYC in HeLa cells. Scale bars, 5 mm. (D) Confocal images of SCOTIN-MYC mutants and endogenous BiP in SCOTIN KO HeLa cells. Scale bars, 5 mm. (E) Quantification data for colocalization of BiP/SCOTIN-MYC in (D) (n = 23–33). The error bars indicate the means ± SDs. See also Figure S3E. (F) Representative immunoblot image showing the protein levels of XBP1s and CHOP in SCOTIN KO HeLa cells expressing the indicated SCOTIN-MYC mutants. (G) Statistical analysis of the band intensities corresponding to the XBP1s and CHOP proteins. The graph of XBP1s shows the means ± SDs of five independent experiments. The graph of CHOP expression presents the means ± SDs of four independent experiments. (H) The production of XBP1s and induction of CHOP in SCOTIN KO HeLa cells transfected with the indicated SCOTIN-MYC mutants were evaluated by western blotting. The asterisks indicate the p values determined by an unpaired two-tailed t test. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001, ****p ˂ 0.0001.

    Journal: Cell reports

    Article Title: Membrane-tethered SCOTIN condensates elicit an endoplasmic reticulum stress response by sequestering luminal BiP.

    doi: 10.1016/j.celrep.2025.115297

    Figure Lengend Snippet: Figure 6. PRD-mediated condensation and BiP sequestration are functionally linked to the induction of ER stress responses (A) Confocal images of SCOTIN-MYC mutants and endogenous BiP in SCOTIN KO HeLa cells. Scale bars, 5 mm. (B) Quantification data for colocalization of BiP/SCOTIN-MYC in (A) (n = 30–119). The error bars indicate the means ± SDs. See also Figure S3D. (C) Confocal images of BiP-EGFP-KDEL mutants and SCOTIN-MYC in HeLa cells. Scale bars, 5 mm. (D) Confocal images of SCOTIN-MYC mutants and endogenous BiP in SCOTIN KO HeLa cells. Scale bars, 5 mm. (E) Quantification data for colocalization of BiP/SCOTIN-MYC in (D) (n = 23–33). The error bars indicate the means ± SDs. See also Figure S3E. (F) Representative immunoblot image showing the protein levels of XBP1s and CHOP in SCOTIN KO HeLa cells expressing the indicated SCOTIN-MYC mutants. (G) Statistical analysis of the band intensities corresponding to the XBP1s and CHOP proteins. The graph of XBP1s shows the means ± SDs of five independent experiments. The graph of CHOP expression presents the means ± SDs of four independent experiments. (H) The production of XBP1s and induction of CHOP in SCOTIN KO HeLa cells transfected with the indicated SCOTIN-MYC mutants were evaluated by western blotting. The asterisks indicate the p values determined by an unpaired two-tailed t test. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001, ****p ˂ 0.0001.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER A549 ATCC Cat# CCL-185 DLD-1 Korean Cell Line Bank Cat# 10221 HCT 116 Korean Cell Line Bank Cat# 10247 HeLa ATCC Cat# CCL-2 HeLa SCOTIN knockout (KO) This study N/A HepG2 ATCC Cat# HB-8065 HT-29 Korean Cell Line Bank Cat# 30038 Huh7 Korean Cell Line Bank Cat# 60104 SW480 Korean Cell Line Bank Cat# 10228 SW620 Korean Cell Line Bank Cat# 60068 U-2 OS Korean Cell Line Bank Cat# 30096 Recombinant DNA SCOTIN This study N/A SCOTIN-TC This study N/A SCOTIN-MYC This study N/A SCOTIN(D150-177)-MYC This study N/A SCOTIN(D29-43)-MYC This study N/A SCOTIN(D44-69)-MYC This study N/A SCOTIN(D70-105)-MYC This study N/A SCOTIN(D44-105)-MYC This study N/A SCOTIN(D29-43,D70-105)-MYC This study N/A SCOTIN(D29-69)-MYC This study N/A SCOTIN(D29-43,D150-177)-MYC This study N/A SCOTIN(D44-69,D150-177)-MYC This study N/A SCOTIN(D70-105,D150-177)-MYC This study N/A SCOTIN(D44-105,D150-177)-MYC This study N/A SCOTIN(D29-43,D70-105,D150-177)-MYC This study N/A SCOTIN(D29-69,D150-177)-MYC This study N/A SCOTIN(DCRD)-MYC This study N/A SCOTIN(DPRD)-MYC This study N/A SCOTIN(DPRD+(150–177))-MYC This study N/A SCOTIN(1–149)-MYC This study N/A SCOTIN(1–177)-MYC This study N/A SCOTIN(DPRD)-FUSN-MYC This study N/A SCOTIN(1–149)-FUSN-MYC This study N/A SCOTIN-APEX2 This study N/A mCherry-Cry2 This study N/A SCOTIN(CRD)-mCherry-Cry2 This study N/A 3xFLAG-ATF6 Chen et al.72 Addgene #11975 IRE1-3F6HGFP Peter Walter Lab (UCSF)29 N/A F-XBP1DDBD-venus Masayuki Miura (University of Tokyo)33 N/A BiP-EGFP-KDEL This study N/A BiP(K294F)-EGFP-KDEL This study N/A BiP(V461F)-EGFP-KDEL This study N/A BiP-(GGGGS)3-mNeonGreen-KDEL This study N/A SCOTIN gRNA/Cas9 knockout plasmid Santa Cruz Cat# sc-408226 SCOTIN HDR plasmid Santa Cruz Cat# sc-408226-HDR Cre Vector Santa Cruz Cat# sc-418923 (Continued on next page) 18 Cell Reports 44, 115297, February 25, 2025

    Techniques: Western Blot, Expressing, Transfection, Two Tailed Test

    Figure 7. Cytosolic condensation of the SCOTIN PRD is essential for the induction of ER stress (A) Schematic of the SCOTIN mutant constructs and chimeric constructs. (B and C) Statistical analysis of F-XBP1DDBD-Venus production in SCOTIN KO HeLa cells expressing the indicated constructs, as determined using flow cy- tometry. See also Figures S4A and S4B. The error bars indicate the means ± SDs from triplicate experiments. (D) Working model of membrane-tethered SCOTIN condensates eliciting an ER stress response by sequestering luminal BiP. The asterisks indicate the p values determined by an unpaired two-tailed t test. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001, ****p ˂ 0.0001.

    Journal: Cell reports

    Article Title: Membrane-tethered SCOTIN condensates elicit an endoplasmic reticulum stress response by sequestering luminal BiP.

    doi: 10.1016/j.celrep.2025.115297

    Figure Lengend Snippet: Figure 7. Cytosolic condensation of the SCOTIN PRD is essential for the induction of ER stress (A) Schematic of the SCOTIN mutant constructs and chimeric constructs. (B and C) Statistical analysis of F-XBP1DDBD-Venus production in SCOTIN KO HeLa cells expressing the indicated constructs, as determined using flow cy- tometry. See also Figures S4A and S4B. The error bars indicate the means ± SDs from triplicate experiments. (D) Working model of membrane-tethered SCOTIN condensates eliciting an ER stress response by sequestering luminal BiP. The asterisks indicate the p values determined by an unpaired two-tailed t test. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001, ****p ˂ 0.0001.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER A549 ATCC Cat# CCL-185 DLD-1 Korean Cell Line Bank Cat# 10221 HCT 116 Korean Cell Line Bank Cat# 10247 HeLa ATCC Cat# CCL-2 HeLa SCOTIN knockout (KO) This study N/A HepG2 ATCC Cat# HB-8065 HT-29 Korean Cell Line Bank Cat# 30038 Huh7 Korean Cell Line Bank Cat# 60104 SW480 Korean Cell Line Bank Cat# 10228 SW620 Korean Cell Line Bank Cat# 60068 U-2 OS Korean Cell Line Bank Cat# 30096 Recombinant DNA SCOTIN This study N/A SCOTIN-TC This study N/A SCOTIN-MYC This study N/A SCOTIN(D150-177)-MYC This study N/A SCOTIN(D29-43)-MYC This study N/A SCOTIN(D44-69)-MYC This study N/A SCOTIN(D70-105)-MYC This study N/A SCOTIN(D44-105)-MYC This study N/A SCOTIN(D29-43,D70-105)-MYC This study N/A SCOTIN(D29-69)-MYC This study N/A SCOTIN(D29-43,D150-177)-MYC This study N/A SCOTIN(D44-69,D150-177)-MYC This study N/A SCOTIN(D70-105,D150-177)-MYC This study N/A SCOTIN(D44-105,D150-177)-MYC This study N/A SCOTIN(D29-43,D70-105,D150-177)-MYC This study N/A SCOTIN(D29-69,D150-177)-MYC This study N/A SCOTIN(DCRD)-MYC This study N/A SCOTIN(DPRD)-MYC This study N/A SCOTIN(DPRD+(150–177))-MYC This study N/A SCOTIN(1–149)-MYC This study N/A SCOTIN(1–177)-MYC This study N/A SCOTIN(DPRD)-FUSN-MYC This study N/A SCOTIN(1–149)-FUSN-MYC This study N/A SCOTIN-APEX2 This study N/A mCherry-Cry2 This study N/A SCOTIN(CRD)-mCherry-Cry2 This study N/A 3xFLAG-ATF6 Chen et al.72 Addgene #11975 IRE1-3F6HGFP Peter Walter Lab (UCSF)29 N/A F-XBP1DDBD-venus Masayuki Miura (University of Tokyo)33 N/A BiP-EGFP-KDEL This study N/A BiP(K294F)-EGFP-KDEL This study N/A BiP(V461F)-EGFP-KDEL This study N/A BiP-(GGGGS)3-mNeonGreen-KDEL This study N/A SCOTIN gRNA/Cas9 knockout plasmid Santa Cruz Cat# sc-408226 SCOTIN HDR plasmid Santa Cruz Cat# sc-408226-HDR Cre Vector Santa Cruz Cat# sc-418923 (Continued on next page) 18 Cell Reports 44, 115297, February 25, 2025

    Techniques: Mutagenesis, Construct, Expressing, Membrane, Two Tailed Test

    Figure 1. SCOTIN inhibits transport from ER to Golgi (A) Schematic illustration of proximal profiling of SCOTIN with BioID. (B) Gene ontology (GO) analysis of proteins identified in the proximal profiling. (C) Schematic illustration of the RUSH reporter system. (D) Cell surface trafficking of E-cadherin-SBP-EGFP was assessed after expression of indicated constructs followed by biotin treatment. (E) Live confocal images of HeLa cells transfected with the E-cadherin-SBP-EGFP with empty or SCOTIN-MYC plasmids during the first 20 min after biotin treatment. (F) (Top) Confocal images of HeLa cells expressing E-cadherin-SBP-EGFP with empty, or SCOTIN-MYC with or without biotin (15 min) followed by immuno- staining with MYC and giantin. (Bottom) The Mander’s coefficient of EGFP overlapping giantin for each cell (n = 6–12). (G) (Top) Confocal images of HeLa cells expressing SBP-EGFP-GPI with empty, or SCOTIN-MYC with or without biotin (7 min) followed by immunostaining with MYC and giantin. (Bottom) The Mander’s coefficient of EGFP overlapping GM130 for each cell (n = 15–17). (H) The trafficking of VSV-Gts045 from ER to Golgi was observed after temperature shift from 40C to 32C for 10 min. Confocal images of HeLa cells after im- munostaining of VSV-G and giantin (top), and Mander’s coefficient of VSV-G overlapping giantin (bottom) were shown (n = 6–8). All graphs are represented as mean ± SEM. All scale bars, 10mm. ***p < 0.001

    Journal: Developmental cell

    Article Title: Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

    doi: 10.1016/j.devcel.2023.08.030

    Figure Lengend Snippet: Figure 1. SCOTIN inhibits transport from ER to Golgi (A) Schematic illustration of proximal profiling of SCOTIN with BioID. (B) Gene ontology (GO) analysis of proteins identified in the proximal profiling. (C) Schematic illustration of the RUSH reporter system. (D) Cell surface trafficking of E-cadherin-SBP-EGFP was assessed after expression of indicated constructs followed by biotin treatment. (E) Live confocal images of HeLa cells transfected with the E-cadherin-SBP-EGFP with empty or SCOTIN-MYC plasmids during the first 20 min after biotin treatment. (F) (Top) Confocal images of HeLa cells expressing E-cadherin-SBP-EGFP with empty, or SCOTIN-MYC with or without biotin (15 min) followed by immuno- staining with MYC and giantin. (Bottom) The Mander’s coefficient of EGFP overlapping giantin for each cell (n = 6–12). (G) (Top) Confocal images of HeLa cells expressing SBP-EGFP-GPI with empty, or SCOTIN-MYC with or without biotin (7 min) followed by immunostaining with MYC and giantin. (Bottom) The Mander’s coefficient of EGFP overlapping GM130 for each cell (n = 15–17). (H) The trafficking of VSV-Gts045 from ER to Golgi was observed after temperature shift from 40C to 32C for 10 min. Confocal images of HeLa cells after im- munostaining of VSV-G and giantin (top), and Mander’s coefficient of VSV-G overlapping giantin (bottom) were shown (n = 6–8). All graphs are represented as mean ± SEM. All scale bars, 10mm. ***p < 0.001

    Article Snippet: SCOTIN KO cell generation with CRISPR/Cas9-mediated genome editing To generate a SCOTIN KO HeLa cell line, a gRNA/Cas9 expression plasmid and a donor plasmid were purchased from Santa Cruz Biotechnology (Table S1).

    Techniques: Expressing, Construct, Transfection, Immunostaining

    Figure 3. Endogenous SCOTIN hijacks Sec31 and interferes ER-to-Golgi transport upon IFN-g stimulation (A) The absence of SCOTIN in the generated SCOTIN KO or KO#2 cells was confirmed by immunoblotting. (B and C) ER-to-Golgi trafficking of the RUSH reporter was compared in WT and SCOTIN KO cells. Cargos are E-cadherin (n = 21–23) in (B) and GPI (n = 19–21) in (C).

    Journal: Developmental cell

    Article Title: Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

    doi: 10.1016/j.devcel.2023.08.030

    Figure Lengend Snippet: Figure 3. Endogenous SCOTIN hijacks Sec31 and interferes ER-to-Golgi transport upon IFN-g stimulation (A) The absence of SCOTIN in the generated SCOTIN KO or KO#2 cells was confirmed by immunoblotting. (B and C) ER-to-Golgi trafficking of the RUSH reporter was compared in WT and SCOTIN KO cells. Cargos are E-cadherin (n = 21–23) in (B) and GPI (n = 19–21) in (C).

    Article Snippet: SCOTIN KO cell generation with CRISPR/Cas9-mediated genome editing To generate a SCOTIN KO HeLa cell line, a gRNA/Cas9 expression plasmid and a donor plasmid were purchased from Santa Cruz Biotechnology (Table S1).

    Techniques: Generated, Western Blot

    Figure 5. SCOTIN condensates are formed with the tubular ER (A–C) Confocal immunofluorescence images of SCOTIN-MYC relative to RTN4 (A), Sec61b (B), or CLIMP63 (C). Scale bars, 10 mm. (D) Airyscan immunofluorescence images of SCOTIN-MYC and RTN4. Representative features are illustrated in the right panel. Scale bars, 10 mm. (E) CLEM images of HeLa cells expressing SCOTIN-mDsRed. The white arrows indicate SCOTIN puncta near the ER membrane. The arrowheads, MT, and v indicate ER, mitochondria, and intracellular vesicles, respectively. Scale bars, 1 mm. (F) CLEM image of HeLa cells expressing SCOTIN(D150–177)-mDsRed. Scale bars, 1 mm. (G) Confocal immunofluorescence images of SCOTIN(D150–177)-mDsRed relative to RTN4. Scale bars, 10 mm.

    Journal: Developmental cell

    Article Title: Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

    doi: 10.1016/j.devcel.2023.08.030

    Figure Lengend Snippet: Figure 5. SCOTIN condensates are formed with the tubular ER (A–C) Confocal immunofluorescence images of SCOTIN-MYC relative to RTN4 (A), Sec61b (B), or CLIMP63 (C). Scale bars, 10 mm. (D) Airyscan immunofluorescence images of SCOTIN-MYC and RTN4. Representative features are illustrated in the right panel. Scale bars, 10 mm. (E) CLEM images of HeLa cells expressing SCOTIN-mDsRed. The white arrows indicate SCOTIN puncta near the ER membrane. The arrowheads, MT, and v indicate ER, mitochondria, and intracellular vesicles, respectively. Scale bars, 1 mm. (F) CLEM image of HeLa cells expressing SCOTIN(D150–177)-mDsRed. Scale bars, 1 mm. (G) Confocal immunofluorescence images of SCOTIN(D150–177)-mDsRed relative to RTN4. Scale bars, 10 mm.

    Article Snippet: SCOTIN KO cell generation with CRISPR/Cas9-mediated genome editing To generate a SCOTIN KO HeLa cell line, a gRNA/Cas9 expression plasmid and a donor plasmid were purchased from Santa Cruz Biotechnology (Table S1).

    Techniques: Expressing, Membrane