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    Addgene inc orai1 coding sequences
    Fig. 1. Generation of Jurkat cell lines stably producing APP. (A) Immunoblot of endogenous APP in Jurkat, 293T/17, and HeLa cells carrying APP-targeting shRNA (APP KD) or control shRNA. (B) qPCR analysis of relative APP mRNA levels in cells presented in (A). Differences from the reference level of 1 set for HeLa control cells were analyzed using one-sample t-test (n ¼ 4 each). Significant differences are marked with asterisks (***p < 0.001). (C) Immunoblot of APP, <t>Orai1,</t> and STIM1 in Jurkat cells stably expressing APP, APPSWE or an empty cassette (control), compared with wildtype HeLa cells. (D) FACS analysis of Y188-labeled Jurkat APP cells and APPSWE cells. Y188-labeled and mock-labeled 293T/17 cells were analyzed in parallel to set a threshold for Y188- positive cells (horizontal line). The x-axis of both histograms shows the Y188 signal normalized to cell size (FL1-H/FSC) on a logarithmic scale.
    Orai1 Coding Sequences, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 22 article reviews
    orai1 coding sequences - by Bioz Stars, 2026-10
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    Images

    1) Product Images from "Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein."

    Article Title: Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2016.08.072

    Fig. 1. Generation of Jurkat cell lines stably producing APP. (A) Immunoblot of endogenous APP in Jurkat, 293T/17, and HeLa cells carrying APP-targeting shRNA (APP KD) or control shRNA. (B) qPCR analysis of relative APP mRNA levels in cells presented in (A). Differences from the reference level of 1 set for HeLa control cells were analyzed using one-sample t-test (n ¼ 4 each). Significant differences are marked with asterisks (***p < 0.001). (C) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing APP, APPSWE or an empty cassette (control), compared with wildtype HeLa cells. (D) FACS analysis of Y188-labeled Jurkat APP cells and APPSWE cells. Y188-labeled and mock-labeled 293T/17 cells were analyzed in parallel to set a threshold for Y188- positive cells (horizontal line). The x-axis of both histograms shows the Y188 signal normalized to cell size (FL1-H/FSC) on a logarithmic scale.
    Figure Legend Snippet: Fig. 1. Generation of Jurkat cell lines stably producing APP. (A) Immunoblot of endogenous APP in Jurkat, 293T/17, and HeLa cells carrying APP-targeting shRNA (APP KD) or control shRNA. (B) qPCR analysis of relative APP mRNA levels in cells presented in (A). Differences from the reference level of 1 set for HeLa control cells were analyzed using one-sample t-test (n ¼ 4 each). Significant differences are marked with asterisks (***p < 0.001). (C) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing APP, APPSWE or an empty cassette (control), compared with wildtype HeLa cells. (D) FACS analysis of Y188-labeled Jurkat APP cells and APPSWE cells. Y188-labeled and mock-labeled 293T/17 cells were analyzed in parallel to set a threshold for Y188- positive cells (horizontal line). The x-axis of both histograms shows the Y188 signal normalized to cell size (FL1-H/FSC) on a logarithmic scale.

    Techniques Used: Stable Transfection, Western Blot, shRNA, Control, Expressing, Labeling

    Fig. 2. Microscopic analysis of SOCE in APP-producing Jurkat cells. (A) SOCE measurements in Fura-4F-loaded Jurkat cells that produced APP (n ¼ 13), APPSWE (n ¼ 12) and control cells with an empty cassette (n ¼ 13). (B) SOCE measurements in Jurkat cells that produced dominant-negative Orai1 (Orai1 DN; n ¼ 5) and control cells (n ¼ 5). Shown are averaged traces from representative experiments and calculated mean values with standard error. Differences from control cells were analyzed using unpaired t-test, and significant dif- ferences are marked with asterisks (***p < 0.001).
    Figure Legend Snippet: Fig. 2. Microscopic analysis of SOCE in APP-producing Jurkat cells. (A) SOCE measurements in Fura-4F-loaded Jurkat cells that produced APP (n ¼ 13), APPSWE (n ¼ 12) and control cells with an empty cassette (n ¼ 13). (B) SOCE measurements in Jurkat cells that produced dominant-negative Orai1 (Orai1 DN; n ¼ 5) and control cells (n ¼ 5). Shown are averaged traces from representative experiments and calculated mean values with standard error. Differences from control cells were analyzed using unpaired t-test, and significant dif- ferences are marked with asterisks (***p < 0.001).

    Techniques Used: Produced, Control, Dominant Negative Mutation

    Fig. 3. Analysis of SOCE in Jurkat cells producing C99 fragments. (A) Immunoblot of small APP fragments (detected with Y188 antibody) in Jurkat cells stably expressing APPSWE, C99 cDNA, or an empty cassette (control) compared with wildtype HeLa cells. DAPT treatment (5 mM for 5 h) is indicated below the gels. (B) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing C99 cDNA, C99IND cDNA, or an empty cassette (control) compared with wildtype HeLa cells. (C) qPCR analysis of ORAI1 and STIM1 mRNA levels in C99- producing Jurkat cells relative to those in Jurkat control cells. Differences were analyzed using one-sample t-test (n ¼ 3 each). (D) SOCE measurements in Fura-4F-loaded Jurkat C99 cells (n ¼ 11), C99IND cells (n ¼ 11) and control cells (n ¼ 12). Traces are from representative experiments. The data were analyzed using unpaired t-test.
    Figure Legend Snippet: Fig. 3. Analysis of SOCE in Jurkat cells producing C99 fragments. (A) Immunoblot of small APP fragments (detected with Y188 antibody) in Jurkat cells stably expressing APPSWE, C99 cDNA, or an empty cassette (control) compared with wildtype HeLa cells. DAPT treatment (5 mM for 5 h) is indicated below the gels. (B) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing C99 cDNA, C99IND cDNA, or an empty cassette (control) compared with wildtype HeLa cells. (C) qPCR analysis of ORAI1 and STIM1 mRNA levels in C99- producing Jurkat cells relative to those in Jurkat control cells. Differences were analyzed using one-sample t-test (n ¼ 3 each). (D) SOCE measurements in Fura-4F-loaded Jurkat C99 cells (n ¼ 11), C99IND cells (n ¼ 11) and control cells (n ¼ 12). Traces are from representative experiments. The data were analyzed using unpaired t-test.

    Techniques Used: Western Blot, Stable Transfection, Expressing, Control

    Fig. 4. Analysis of SOCE in HeLa cells depleted of APP. (A) SOCE measurements in Fura-2-loaded HeLa cells stably expressing APP-targeting shRNA (APP KD; n ¼ 8), ORAI1-targeting shRNA (ORAI1 KD; n ¼ 3) and control shRNA (control; n ¼ 8). The stores were depleted with 20 mM CPA in Ca2þ-free buffer containing 0.5 mM EGTA for 400 s, and SOCE was initiated by the re-addition of 2 mM Ca2þ. Shown are averaged (black) and individual (gray) SOCE traces from representative experiments. The data were analyzed using unpaired t- test, and significant differences from controls are marked with asterisks (***p < 0.001). (B) qPCR analysis of ORAI1 and STIM1 mRNAs in cells presented in (A). Differences from the reference level of 1 set for controls were analyzed using one-sample t-test (n ¼ 3 each). Statistical significance is marked with asterisks (***p < 0.001).
    Figure Legend Snippet: Fig. 4. Analysis of SOCE in HeLa cells depleted of APP. (A) SOCE measurements in Fura-2-loaded HeLa cells stably expressing APP-targeting shRNA (APP KD; n ¼ 8), ORAI1-targeting shRNA (ORAI1 KD; n ¼ 3) and control shRNA (control; n ¼ 8). The stores were depleted with 20 mM CPA in Ca2þ-free buffer containing 0.5 mM EGTA for 400 s, and SOCE was initiated by the re-addition of 2 mM Ca2þ. Shown are averaged (black) and individual (gray) SOCE traces from representative experiments. The data were analyzed using unpaired t- test, and significant differences from controls are marked with asterisks (***p < 0.001). (B) qPCR analysis of ORAI1 and STIM1 mRNAs in cells presented in (A). Differences from the reference level of 1 set for controls were analyzed using one-sample t-test (n ¼ 3 each). Statistical significance is marked with asterisks (***p < 0.001).

    Techniques Used: Stable Transfection, Expressing, shRNA, Control

    Related Articles

    Transfection:

    Article Title: STING orchestrates the neuronal inflammatory stress response in multiple sclerosis.
    Article Snippet: We acquired images with a confocal LSM 700 laser scanning confocal microscope (Zeiss) every 0.48 seconds with a 203magnification in an imaging chamber maintaining 37 C and 5% CO2. .. To visualize ER-calcium or calcium flux through ORAI1 we transfected ERGcaMP6-210 (ER-GcaMP6-210 was a gift from Timothy Ryan (Addgene plasmid #86919; http://n2t.net/addgene:86919; RRID:Addgene_86919))93 or Orai1-GcaMP6f (Orai1-GcaMP6f was a gift from Michael Cahalan (Addgene plasmid # 73564; http://n2t. net/addgene:73564; RRID:Addgene_73564))94 using lipofectamine at 3 d.i.v. (see above for detailed description of lipofectamine transfection). .. We acquired images with a confocal LSM 700 laser scanning confocal microscope (Zeiss) every 0.48 seconds with a 203magnification in an imaging chamber maintaining 37 C and 5% CO2.

    Article Title: Water in peripheral TM-interfaces of Orai1-channels triggers pore opening
    Article Snippet: Transient transfection of human embryonic kidney (HEK) 293 cells was performed using the TransFectin Lipid Reagent (Bio-Rad) (New England Biolabs) . .. For each transfection, Orai1 plasmids together with STIM1 plasmids, R-Geco1.2 (purchased from Addgene ) or NFAT were used at a 1:1 ratio. ..

    Plasmid Preparation:

    Article Title: STING orchestrates the neuronal inflammatory stress response in multiple sclerosis.
    Article Snippet: We acquired images with a confocal LSM 700 laser scanning confocal microscope (Zeiss) every 0.48 seconds with a 203magnification in an imaging chamber maintaining 37 C and 5% CO2. .. To visualize ER-calcium or calcium flux through ORAI1 we transfected ERGcaMP6-210 (ER-GcaMP6-210 was a gift from Timothy Ryan (Addgene plasmid #86919; http://n2t.net/addgene:86919; RRID:Addgene_86919))93 or Orai1-GcaMP6f (Orai1-GcaMP6f was a gift from Michael Cahalan (Addgene plasmid # 73564; http://n2t. net/addgene:73564; RRID:Addgene_73564))94 using lipofectamine at 3 d.i.v. (see above for detailed description of lipofectamine transfection). .. We acquired images with a confocal LSM 700 laser scanning confocal microscope (Zeiss) every 0.48 seconds with a 203magnification in an imaging chamber maintaining 37 C and 5% CO2.

    Article Title: SPLUNC1 is a negative regulator of the Orai1 Ca 2+ channel
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    Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry.
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    Clone Assay:

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    Polymerase Chain Reaction:

    Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry.
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    Purification:

    Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals and peptides RPMI-1640 Gibco Cat# 11875093 Fetal Bovine Serum, heat inactivated Gibco Cat# 16140-071 Penicillin/Streptomycin Gibco Cat# 15140122 DMEM Gibco Cat# 10566016 PBS Gibco Cat# 10010023 TransIT-Lenti Transfection Reagent Mirus Bio Cat# MIR 6603 FR180204 (ERK Inhibitor II) Cayman Cat# 15544 DMSO Alfa Aesar Cat# 42780 eBioscience TM Protein Transport Inhibitor Cocktail Thermo Fisher Cat# 00-4980-93 IC Fixation Buffer Thermo Fisher Cat# 00-8222-49 Permeabilization Buffer Thermo Fisher Cat# 00-8333-56 eBioscience TM Flow Cytometry Staining Buffer Thermo Fisher Cat# 00-4222-26 Proteinase K Zymo Cat# D3001-2-5 Polybrene MilliporeSigma Cat# TR-1003 Puromycin MedChemExpress Cat# HY-B1743 Doxycycline Thermo Fisher Cat# J60579.22 Propidium iodide MilliporeSigma Cat# P4170 Paraformaldehyde, methanol-free, 16% Electron Microscopy Sciences Cat# 15710 Triton X-100 (DNAse, RNAse, protease free) Acros Organics Cat# 327371000 RIPA lysis buffer Thermo Fisher Cat# 89900 SRC-selective tyrosine kinase inhibitor PP1 MilliporeSigma Cat# 5678091MG Ammonium Bicarbonate Thermo Fisher Cat# 393210010 Urea Thermo Fisher Cat# 29700 TCEP Thermo Fisher Cat# PG82080 Chloro-acetamide Thermo Fisher Cat# A15238.30 Trypsin/EDTA Gibco Cat# 25300054 Hydroxylamine Thermo Fisher Cat# A15398.0I Acetonitrile Thermo Fisher Cat# 047138.M1 Formic acid Thermo Fisher Cat# 28905 Sorafenib (Bay 43–9006) MedChemExpress Cat# HY-10201 Ulixertinib (BVD-523) MedChemExpress Cat# HY-15816 Zegocractin (CM-4620) MedChemExpress Cat# HY-101942 Critical commercial assays EasySep TM Direct Human PBMC Isolation Kit STEMCELL Technologies Cat# 19654 Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x genomics Cat# PN-1000283 Chromium Fixed RNA Kit, Human Transcriptome 10x genomics Cat# PN-1000476 Dead cell removal kit Miltenyi Biotec Cat# 130-090-101 Quick-DNA/RNA Miniprep Kit Zymo Cat# D7005T Dead cell apoptosis kit, Alexa 488 w/Annexin V PI Invitrogen Cat# V13241 RNeasy® Mini Kit Qiagen Cat# 74104 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Cat# 4368814 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Nextera Library Prep Kit Illumina Cat# FC-131-1096 (Continued on next page) Cell Reports 45, 116903, February 24, 2026 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER MinElute PCR purification Qiagen Cat# 28004 KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 CUTANA TM CUT&RUN Library Prep Kit EpiCypher Cat# 14-1001 Qubit 1X dsDNA HS Assay Kit Thermo Fisher Cat# Q33230 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Duolink® In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Oligonucleotides qPCR primers for IL8, GAPDH, and RN18S1: see Table S2 This study N/A gRNA sequences for STIM1, ORAI1, SPTAN1, and RAF1 knockouts: see Table S3 This study N/A Plasmid lentiCRISPRv2 Addgene Cat# 73179-LV Lenti-iCas9-neo Addgene Cat# 85400 Lenti-multi-Guide plasmid Addgene Cat# 85401 pCDH-CMV-Nluc Addgene Cat#73038 pLenti CMV GFP Puro Addgene Cat# 658-5 pcDNA3-Venus-173-N-ORAI1 Addgene Cat# 87618 pcDNA3.1-STIM1-Venus-173-C Addgene Cat# 87619 pCDH-CMV-Nluc Addgene Cat# 73038 Biological samples Human peripheral blood mononuclear cells (PBMCs) This study N/A THP-1 cells ATCC TIB-202 Lenti-X 293T Takara Bio Cat# 632180 Human CD14 + primary monocytes Bloodworks Bio 4570–61 Antibodies IL8 antibody Invitrogen Cat# 12808842; RRID: AB_2784632 Anti-Ras antibody Abcam Cat# AB108602; RRID: AB_10891004 Raf1 antibody Abcam Cat# AB137435; RRID: AB_3674177 Goat anti-Mouse Alexa Fluor 488 Life Technologies Cat# A32723; RRID: AB_2633275 Goat anti-Rabbit, Alexa Fluor 594 Life Technologies Cat# A32740; RRID: AB_2762824 Anti-Spectrin alpha chain (nonerythroid) Antibody MilliporeSigma Cat# MAB1622; RRID: AB_94295 p-ERK1/2 antibody Cell Signaling Cat# 9101; RRID: AB_331646 ERK1/2 antibody Cell Signaling Cat# 4695; RRID: AB_390779 IRDye 680RD Goat anti-Mouse Li-Cor Cat# 926-68070; RRID: AB_10956588 IRDye 800CW Goat anti-Rabbit Li-Cor Cat# 926-32211; RRID: AB_621843 c-Jun Antibody Thermo Fisher Cat# MA5-15881; RRID: AB_11153582 β-actin Santa Cruz Cat# SC-47778; RRID: AB_626632 Deposited data snRNA-seq/snATAC-seq, ATAC, bulk-RNA, CUT&RUN data This paper NCBI GEO: GSE262146 CRISPRi screen data: See Data S1 This paper NCBI GEO: GSE213184 scRNA-seq data of drug treatment This paper NCBI GEO: GSE291575 Phospho-proteomics: See Data S2 This paper N/A Software and algorithms R 4.2.3 CRAN project https://www.r-project.org/ Seurat R package Version: 4.3.0.1 (Continued on next page) 16 Cell Reports 45, 116903, February 24, 2026 ..

    Library Quantification:

    Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals and peptides RPMI-1640 Gibco Cat# 11875093 Fetal Bovine Serum, heat inactivated Gibco Cat# 16140-071 Penicillin/Streptomycin Gibco Cat# 15140122 DMEM Gibco Cat# 10566016 PBS Gibco Cat# 10010023 TransIT-Lenti Transfection Reagent Mirus Bio Cat# MIR 6603 FR180204 (ERK Inhibitor II) Cayman Cat# 15544 DMSO Alfa Aesar Cat# 42780 eBioscience TM Protein Transport Inhibitor Cocktail Thermo Fisher Cat# 00-4980-93 IC Fixation Buffer Thermo Fisher Cat# 00-8222-49 Permeabilization Buffer Thermo Fisher Cat# 00-8333-56 eBioscience TM Flow Cytometry Staining Buffer Thermo Fisher Cat# 00-4222-26 Proteinase K Zymo Cat# D3001-2-5 Polybrene MilliporeSigma Cat# TR-1003 Puromycin MedChemExpress Cat# HY-B1743 Doxycycline Thermo Fisher Cat# J60579.22 Propidium iodide MilliporeSigma Cat# P4170 Paraformaldehyde, methanol-free, 16% Electron Microscopy Sciences Cat# 15710 Triton X-100 (DNAse, RNAse, protease free) Acros Organics Cat# 327371000 RIPA lysis buffer Thermo Fisher Cat# 89900 SRC-selective tyrosine kinase inhibitor PP1 MilliporeSigma Cat# 5678091MG Ammonium Bicarbonate Thermo Fisher Cat# 393210010 Urea Thermo Fisher Cat# 29700 TCEP Thermo Fisher Cat# PG82080 Chloro-acetamide Thermo Fisher Cat# A15238.30 Trypsin/EDTA Gibco Cat# 25300054 Hydroxylamine Thermo Fisher Cat# A15398.0I Acetonitrile Thermo Fisher Cat# 047138.M1 Formic acid Thermo Fisher Cat# 28905 Sorafenib (Bay 43–9006) MedChemExpress Cat# HY-10201 Ulixertinib (BVD-523) MedChemExpress Cat# HY-15816 Zegocractin (CM-4620) MedChemExpress Cat# HY-101942 Critical commercial assays EasySep TM Direct Human PBMC Isolation Kit STEMCELL Technologies Cat# 19654 Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x genomics Cat# PN-1000283 Chromium Fixed RNA Kit, Human Transcriptome 10x genomics Cat# PN-1000476 Dead cell removal kit Miltenyi Biotec Cat# 130-090-101 Quick-DNA/RNA Miniprep Kit Zymo Cat# D7005T Dead cell apoptosis kit, Alexa 488 w/Annexin V PI Invitrogen Cat# V13241 RNeasy® Mini Kit Qiagen Cat# 74104 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Cat# 4368814 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Nextera Library Prep Kit Illumina Cat# FC-131-1096 (Continued on next page) Cell Reports 45, 116903, February 24, 2026 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER MinElute PCR purification Qiagen Cat# 28004 KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 CUTANA TM CUT&RUN Library Prep Kit EpiCypher Cat# 14-1001 Qubit 1X dsDNA HS Assay Kit Thermo Fisher Cat# Q33230 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Duolink® In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Oligonucleotides qPCR primers for IL8, GAPDH, and RN18S1: see Table S2 This study N/A gRNA sequences for STIM1, ORAI1, SPTAN1, and RAF1 knockouts: see Table S3 This study N/A Plasmid lentiCRISPRv2 Addgene Cat# 73179-LV Lenti-iCas9-neo Addgene Cat# 85400 Lenti-multi-Guide plasmid Addgene Cat# 85401 pCDH-CMV-Nluc Addgene Cat#73038 pLenti CMV GFP Puro Addgene Cat# 658-5 pcDNA3-Venus-173-N-ORAI1 Addgene Cat# 87618 pcDNA3.1-STIM1-Venus-173-C Addgene Cat# 87619 pCDH-CMV-Nluc Addgene Cat# 73038 Biological samples Human peripheral blood mononuclear cells (PBMCs) This study N/A THP-1 cells ATCC TIB-202 Lenti-X 293T Takara Bio Cat# 632180 Human CD14 + primary monocytes Bloodworks Bio 4570–61 Antibodies IL8 antibody Invitrogen Cat# 12808842; RRID: AB_2784632 Anti-Ras antibody Abcam Cat# AB108602; RRID: AB_10891004 Raf1 antibody Abcam Cat# AB137435; RRID: AB_3674177 Goat anti-Mouse Alexa Fluor 488 Life Technologies Cat# A32723; RRID: AB_2633275 Goat anti-Rabbit, Alexa Fluor 594 Life Technologies Cat# A32740; RRID: AB_2762824 Anti-Spectrin alpha chain (nonerythroid) Antibody MilliporeSigma Cat# MAB1622; RRID: AB_94295 p-ERK1/2 antibody Cell Signaling Cat# 9101; RRID: AB_331646 ERK1/2 antibody Cell Signaling Cat# 4695; RRID: AB_390779 IRDye 680RD Goat anti-Mouse Li-Cor Cat# 926-68070; RRID: AB_10956588 IRDye 800CW Goat anti-Rabbit Li-Cor Cat# 926-32211; RRID: AB_621843 c-Jun Antibody Thermo Fisher Cat# MA5-15881; RRID: AB_11153582 β-actin Santa Cruz Cat# SC-47778; RRID: AB_626632 Deposited data snRNA-seq/snATAC-seq, ATAC, bulk-RNA, CUT&RUN data This paper NCBI GEO: GSE262146 CRISPRi screen data: See Data S1 This paper NCBI GEO: GSE213184 scRNA-seq data of drug treatment This paper NCBI GEO: GSE291575 Phospho-proteomics: See Data S2 This paper N/A Software and algorithms R 4.2.3 CRAN project https://www.r-project.org/ Seurat R package Version: 4.3.0.1 (Continued on next page) 16 Cell Reports 45, 116903, February 24, 2026 ..

    BIA-KA:

    Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals and peptides RPMI-1640 Gibco Cat# 11875093 Fetal Bovine Serum, heat inactivated Gibco Cat# 16140-071 Penicillin/Streptomycin Gibco Cat# 15140122 DMEM Gibco Cat# 10566016 PBS Gibco Cat# 10010023 TransIT-Lenti Transfection Reagent Mirus Bio Cat# MIR 6603 FR180204 (ERK Inhibitor II) Cayman Cat# 15544 DMSO Alfa Aesar Cat# 42780 eBioscience TM Protein Transport Inhibitor Cocktail Thermo Fisher Cat# 00-4980-93 IC Fixation Buffer Thermo Fisher Cat# 00-8222-49 Permeabilization Buffer Thermo Fisher Cat# 00-8333-56 eBioscience TM Flow Cytometry Staining Buffer Thermo Fisher Cat# 00-4222-26 Proteinase K Zymo Cat# D3001-2-5 Polybrene MilliporeSigma Cat# TR-1003 Puromycin MedChemExpress Cat# HY-B1743 Doxycycline Thermo Fisher Cat# J60579.22 Propidium iodide MilliporeSigma Cat# P4170 Paraformaldehyde, methanol-free, 16% Electron Microscopy Sciences Cat# 15710 Triton X-100 (DNAse, RNAse, protease free) Acros Organics Cat# 327371000 RIPA lysis buffer Thermo Fisher Cat# 89900 SRC-selective tyrosine kinase inhibitor PP1 MilliporeSigma Cat# 5678091MG Ammonium Bicarbonate Thermo Fisher Cat# 393210010 Urea Thermo Fisher Cat# 29700 TCEP Thermo Fisher Cat# PG82080 Chloro-acetamide Thermo Fisher Cat# A15238.30 Trypsin/EDTA Gibco Cat# 25300054 Hydroxylamine Thermo Fisher Cat# A15398.0I Acetonitrile Thermo Fisher Cat# 047138.M1 Formic acid Thermo Fisher Cat# 28905 Sorafenib (Bay 43–9006) MedChemExpress Cat# HY-10201 Ulixertinib (BVD-523) MedChemExpress Cat# HY-15816 Zegocractin (CM-4620) MedChemExpress Cat# HY-101942 Critical commercial assays EasySep TM Direct Human PBMC Isolation Kit STEMCELL Technologies Cat# 19654 Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x genomics Cat# PN-1000283 Chromium Fixed RNA Kit, Human Transcriptome 10x genomics Cat# PN-1000476 Dead cell removal kit Miltenyi Biotec Cat# 130-090-101 Quick-DNA/RNA Miniprep Kit Zymo Cat# D7005T Dead cell apoptosis kit, Alexa 488 w/Annexin V PI Invitrogen Cat# V13241 RNeasy® Mini Kit Qiagen Cat# 74104 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Cat# 4368814 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Nextera Library Prep Kit Illumina Cat# FC-131-1096 (Continued on next page) Cell Reports 45, 116903, February 24, 2026 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER MinElute PCR purification Qiagen Cat# 28004 KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 CUTANA TM CUT&RUN Library Prep Kit EpiCypher Cat# 14-1001 Qubit 1X dsDNA HS Assay Kit Thermo Fisher Cat# Q33230 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Duolink® In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Oligonucleotides qPCR primers for IL8, GAPDH, and RN18S1: see Table S2 This study N/A gRNA sequences for STIM1, ORAI1, SPTAN1, and RAF1 knockouts: see Table S3 This study N/A Plasmid lentiCRISPRv2 Addgene Cat# 73179-LV Lenti-iCas9-neo Addgene Cat# 85400 Lenti-multi-Guide plasmid Addgene Cat# 85401 pCDH-CMV-Nluc Addgene Cat#73038 pLenti CMV GFP Puro Addgene Cat# 658-5 pcDNA3-Venus-173-N-ORAI1 Addgene Cat# 87618 pcDNA3.1-STIM1-Venus-173-C Addgene Cat# 87619 pCDH-CMV-Nluc Addgene Cat# 73038 Biological samples Human peripheral blood mononuclear cells (PBMCs) This study N/A THP-1 cells ATCC TIB-202 Lenti-X 293T Takara Bio Cat# 632180 Human CD14 + primary monocytes Bloodworks Bio 4570–61 Antibodies IL8 antibody Invitrogen Cat# 12808842; RRID: AB_2784632 Anti-Ras antibody Abcam Cat# AB108602; RRID: AB_10891004 Raf1 antibody Abcam Cat# AB137435; RRID: AB_3674177 Goat anti-Mouse Alexa Fluor 488 Life Technologies Cat# A32723; RRID: AB_2633275 Goat anti-Rabbit, Alexa Fluor 594 Life Technologies Cat# A32740; RRID: AB_2762824 Anti-Spectrin alpha chain (nonerythroid) Antibody MilliporeSigma Cat# MAB1622; RRID: AB_94295 p-ERK1/2 antibody Cell Signaling Cat# 9101; RRID: AB_331646 ERK1/2 antibody Cell Signaling Cat# 4695; RRID: AB_390779 IRDye 680RD Goat anti-Mouse Li-Cor Cat# 926-68070; RRID: AB_10956588 IRDye 800CW Goat anti-Rabbit Li-Cor Cat# 926-32211; RRID: AB_621843 c-Jun Antibody Thermo Fisher Cat# MA5-15881; RRID: AB_11153582 β-actin Santa Cruz Cat# SC-47778; RRID: AB_626632 Deposited data snRNA-seq/snATAC-seq, ATAC, bulk-RNA, CUT&RUN data This paper NCBI GEO: GSE262146 CRISPRi screen data: See Data S1 This paper NCBI GEO: GSE213184 scRNA-seq data of drug treatment This paper NCBI GEO: GSE291575 Phospho-proteomics: See Data S2 This paper N/A Software and algorithms R 4.2.3 CRAN project https://www.r-project.org/ Seurat R package Version: 4.3.0.1 (Continued on next page) 16 Cell Reports 45, 116903, February 24, 2026 ..

    In Situ:

    Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals and peptides RPMI-1640 Gibco Cat# 11875093 Fetal Bovine Serum, heat inactivated Gibco Cat# 16140-071 Penicillin/Streptomycin Gibco Cat# 15140122 DMEM Gibco Cat# 10566016 PBS Gibco Cat# 10010023 TransIT-Lenti Transfection Reagent Mirus Bio Cat# MIR 6603 FR180204 (ERK Inhibitor II) Cayman Cat# 15544 DMSO Alfa Aesar Cat# 42780 eBioscience TM Protein Transport Inhibitor Cocktail Thermo Fisher Cat# 00-4980-93 IC Fixation Buffer Thermo Fisher Cat# 00-8222-49 Permeabilization Buffer Thermo Fisher Cat# 00-8333-56 eBioscience TM Flow Cytometry Staining Buffer Thermo Fisher Cat# 00-4222-26 Proteinase K Zymo Cat# D3001-2-5 Polybrene MilliporeSigma Cat# TR-1003 Puromycin MedChemExpress Cat# HY-B1743 Doxycycline Thermo Fisher Cat# J60579.22 Propidium iodide MilliporeSigma Cat# P4170 Paraformaldehyde, methanol-free, 16% Electron Microscopy Sciences Cat# 15710 Triton X-100 (DNAse, RNAse, protease free) Acros Organics Cat# 327371000 RIPA lysis buffer Thermo Fisher Cat# 89900 SRC-selective tyrosine kinase inhibitor PP1 MilliporeSigma Cat# 5678091MG Ammonium Bicarbonate Thermo Fisher Cat# 393210010 Urea Thermo Fisher Cat# 29700 TCEP Thermo Fisher Cat# PG82080 Chloro-acetamide Thermo Fisher Cat# A15238.30 Trypsin/EDTA Gibco Cat# 25300054 Hydroxylamine Thermo Fisher Cat# A15398.0I Acetonitrile Thermo Fisher Cat# 047138.M1 Formic acid Thermo Fisher Cat# 28905 Sorafenib (Bay 43–9006) MedChemExpress Cat# HY-10201 Ulixertinib (BVD-523) MedChemExpress Cat# HY-15816 Zegocractin (CM-4620) MedChemExpress Cat# HY-101942 Critical commercial assays EasySep TM Direct Human PBMC Isolation Kit STEMCELL Technologies Cat# 19654 Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x genomics Cat# PN-1000283 Chromium Fixed RNA Kit, Human Transcriptome 10x genomics Cat# PN-1000476 Dead cell removal kit Miltenyi Biotec Cat# 130-090-101 Quick-DNA/RNA Miniprep Kit Zymo Cat# D7005T Dead cell apoptosis kit, Alexa 488 w/Annexin V PI Invitrogen Cat# V13241 RNeasy® Mini Kit Qiagen Cat# 74104 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Cat# 4368814 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Nextera Library Prep Kit Illumina Cat# FC-131-1096 (Continued on next page) Cell Reports 45, 116903, February 24, 2026 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER MinElute PCR purification Qiagen Cat# 28004 KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 CUTANA TM CUT&RUN Library Prep Kit EpiCypher Cat# 14-1001 Qubit 1X dsDNA HS Assay Kit Thermo Fisher Cat# Q33230 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Duolink® In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Oligonucleotides qPCR primers for IL8, GAPDH, and RN18S1: see Table S2 This study N/A gRNA sequences for STIM1, ORAI1, SPTAN1, and RAF1 knockouts: see Table S3 This study N/A Plasmid lentiCRISPRv2 Addgene Cat# 73179-LV Lenti-iCas9-neo Addgene Cat# 85400 Lenti-multi-Guide plasmid Addgene Cat# 85401 pCDH-CMV-Nluc Addgene Cat#73038 pLenti CMV GFP Puro Addgene Cat# 658-5 pcDNA3-Venus-173-N-ORAI1 Addgene Cat# 87618 pcDNA3.1-STIM1-Venus-173-C Addgene Cat# 87619 pCDH-CMV-Nluc Addgene Cat# 73038 Biological samples Human peripheral blood mononuclear cells (PBMCs) This study N/A THP-1 cells ATCC TIB-202 Lenti-X 293T Takara Bio Cat# 632180 Human CD14 + primary monocytes Bloodworks Bio 4570–61 Antibodies IL8 antibody Invitrogen Cat# 12808842; RRID: AB_2784632 Anti-Ras antibody Abcam Cat# AB108602; RRID: AB_10891004 Raf1 antibody Abcam Cat# AB137435; RRID: AB_3674177 Goat anti-Mouse Alexa Fluor 488 Life Technologies Cat# A32723; RRID: AB_2633275 Goat anti-Rabbit, Alexa Fluor 594 Life Technologies Cat# A32740; RRID: AB_2762824 Anti-Spectrin alpha chain (nonerythroid) Antibody MilliporeSigma Cat# MAB1622; RRID: AB_94295 p-ERK1/2 antibody Cell Signaling Cat# 9101; RRID: AB_331646 ERK1/2 antibody Cell Signaling Cat# 4695; RRID: AB_390779 IRDye 680RD Goat anti-Mouse Li-Cor Cat# 926-68070; RRID: AB_10956588 IRDye 800CW Goat anti-Rabbit Li-Cor Cat# 926-32211; RRID: AB_621843 c-Jun Antibody Thermo Fisher Cat# MA5-15881; RRID: AB_11153582 β-actin Santa Cruz Cat# SC-47778; RRID: AB_626632 Deposited data snRNA-seq/snATAC-seq, ATAC, bulk-RNA, CUT&RUN data This paper NCBI GEO: GSE262146 CRISPRi screen data: See Data S1 This paper NCBI GEO: GSE213184 scRNA-seq data of drug treatment This paper NCBI GEO: GSE291575 Phospho-proteomics: See Data S2 This paper N/A Software and algorithms R 4.2.3 CRAN project https://www.r-project.org/ Seurat R package Version: 4.3.0.1 (Continued on next page) 16 Cell Reports 45, 116903, February 24, 2026 ..

    Real-time Polymerase Chain Reaction:

    Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals and peptides RPMI-1640 Gibco Cat# 11875093 Fetal Bovine Serum, heat inactivated Gibco Cat# 16140-071 Penicillin/Streptomycin Gibco Cat# 15140122 DMEM Gibco Cat# 10566016 PBS Gibco Cat# 10010023 TransIT-Lenti Transfection Reagent Mirus Bio Cat# MIR 6603 FR180204 (ERK Inhibitor II) Cayman Cat# 15544 DMSO Alfa Aesar Cat# 42780 eBioscience TM Protein Transport Inhibitor Cocktail Thermo Fisher Cat# 00-4980-93 IC Fixation Buffer Thermo Fisher Cat# 00-8222-49 Permeabilization Buffer Thermo Fisher Cat# 00-8333-56 eBioscience TM Flow Cytometry Staining Buffer Thermo Fisher Cat# 00-4222-26 Proteinase K Zymo Cat# D3001-2-5 Polybrene MilliporeSigma Cat# TR-1003 Puromycin MedChemExpress Cat# HY-B1743 Doxycycline Thermo Fisher Cat# J60579.22 Propidium iodide MilliporeSigma Cat# P4170 Paraformaldehyde, methanol-free, 16% Electron Microscopy Sciences Cat# 15710 Triton X-100 (DNAse, RNAse, protease free) Acros Organics Cat# 327371000 RIPA lysis buffer Thermo Fisher Cat# 89900 SRC-selective tyrosine kinase inhibitor PP1 MilliporeSigma Cat# 5678091MG Ammonium Bicarbonate Thermo Fisher Cat# 393210010 Urea Thermo Fisher Cat# 29700 TCEP Thermo Fisher Cat# PG82080 Chloro-acetamide Thermo Fisher Cat# A15238.30 Trypsin/EDTA Gibco Cat# 25300054 Hydroxylamine Thermo Fisher Cat# A15398.0I Acetonitrile Thermo Fisher Cat# 047138.M1 Formic acid Thermo Fisher Cat# 28905 Sorafenib (Bay 43–9006) MedChemExpress Cat# HY-10201 Ulixertinib (BVD-523) MedChemExpress Cat# HY-15816 Zegocractin (CM-4620) MedChemExpress Cat# HY-101942 Critical commercial assays EasySep TM Direct Human PBMC Isolation Kit STEMCELL Technologies Cat# 19654 Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x genomics Cat# PN-1000283 Chromium Fixed RNA Kit, Human Transcriptome 10x genomics Cat# PN-1000476 Dead cell removal kit Miltenyi Biotec Cat# 130-090-101 Quick-DNA/RNA Miniprep Kit Zymo Cat# D7005T Dead cell apoptosis kit, Alexa 488 w/Annexin V PI Invitrogen Cat# V13241 RNeasy® Mini Kit Qiagen Cat# 74104 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Cat# 4368814 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Nextera Library Prep Kit Illumina Cat# FC-131-1096 (Continued on next page) Cell Reports 45, 116903, February 24, 2026 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER MinElute PCR purification Qiagen Cat# 28004 KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 CUTANA TM CUT&RUN Library Prep Kit EpiCypher Cat# 14-1001 Qubit 1X dsDNA HS Assay Kit Thermo Fisher Cat# Q33230 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Duolink® In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Oligonucleotides qPCR primers for IL8, GAPDH, and RN18S1: see Table S2 This study N/A gRNA sequences for STIM1, ORAI1, SPTAN1, and RAF1 knockouts: see Table S3 This study N/A Plasmid lentiCRISPRv2 Addgene Cat# 73179-LV Lenti-iCas9-neo Addgene Cat# 85400 Lenti-multi-Guide plasmid Addgene Cat# 85401 pCDH-CMV-Nluc Addgene Cat#73038 pLenti CMV GFP Puro Addgene Cat# 658-5 pcDNA3-Venus-173-N-ORAI1 Addgene Cat# 87618 pcDNA3.1-STIM1-Venus-173-C Addgene Cat# 87619 pCDH-CMV-Nluc Addgene Cat# 73038 Biological samples Human peripheral blood mononuclear cells (PBMCs) This study N/A THP-1 cells ATCC TIB-202 Lenti-X 293T Takara Bio Cat# 632180 Human CD14 + primary monocytes Bloodworks Bio 4570–61 Antibodies IL8 antibody Invitrogen Cat# 12808842; RRID: AB_2784632 Anti-Ras antibody Abcam Cat# AB108602; RRID: AB_10891004 Raf1 antibody Abcam Cat# AB137435; RRID: AB_3674177 Goat anti-Mouse Alexa Fluor 488 Life Technologies Cat# A32723; RRID: AB_2633275 Goat anti-Rabbit, Alexa Fluor 594 Life Technologies Cat# A32740; RRID: AB_2762824 Anti-Spectrin alpha chain (nonerythroid) Antibody MilliporeSigma Cat# MAB1622; RRID: AB_94295 p-ERK1/2 antibody Cell Signaling Cat# 9101; RRID: AB_331646 ERK1/2 antibody Cell Signaling Cat# 4695; RRID: AB_390779 IRDye 680RD Goat anti-Mouse Li-Cor Cat# 926-68070; RRID: AB_10956588 IRDye 800CW Goat anti-Rabbit Li-Cor Cat# 926-32211; RRID: AB_621843 c-Jun Antibody Thermo Fisher Cat# MA5-15881; RRID: AB_11153582 β-actin Santa Cruz Cat# SC-47778; RRID: AB_626632 Deposited data snRNA-seq/snATAC-seq, ATAC, bulk-RNA, CUT&RUN data This paper NCBI GEO: GSE262146 CRISPRi screen data: See Data S1 This paper NCBI GEO: GSE213184 scRNA-seq data of drug treatment This paper NCBI GEO: GSE291575 Phospho-proteomics: See Data S2 This paper N/A Software and algorithms R 4.2.3 CRAN project https://www.r-project.org/ Seurat R package Version: 4.3.0.1 (Continued on next page) 16 Cell Reports 45, 116903, February 24, 2026 ..

    Phospho-proteomics:

    Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals and peptides RPMI-1640 Gibco Cat# 11875093 Fetal Bovine Serum, heat inactivated Gibco Cat# 16140-071 Penicillin/Streptomycin Gibco Cat# 15140122 DMEM Gibco Cat# 10566016 PBS Gibco Cat# 10010023 TransIT-Lenti Transfection Reagent Mirus Bio Cat# MIR 6603 FR180204 (ERK Inhibitor II) Cayman Cat# 15544 DMSO Alfa Aesar Cat# 42780 eBioscience TM Protein Transport Inhibitor Cocktail Thermo Fisher Cat# 00-4980-93 IC Fixation Buffer Thermo Fisher Cat# 00-8222-49 Permeabilization Buffer Thermo Fisher Cat# 00-8333-56 eBioscience TM Flow Cytometry Staining Buffer Thermo Fisher Cat# 00-4222-26 Proteinase K Zymo Cat# D3001-2-5 Polybrene MilliporeSigma Cat# TR-1003 Puromycin MedChemExpress Cat# HY-B1743 Doxycycline Thermo Fisher Cat# J60579.22 Propidium iodide MilliporeSigma Cat# P4170 Paraformaldehyde, methanol-free, 16% Electron Microscopy Sciences Cat# 15710 Triton X-100 (DNAse, RNAse, protease free) Acros Organics Cat# 327371000 RIPA lysis buffer Thermo Fisher Cat# 89900 SRC-selective tyrosine kinase inhibitor PP1 MilliporeSigma Cat# 5678091MG Ammonium Bicarbonate Thermo Fisher Cat# 393210010 Urea Thermo Fisher Cat# 29700 TCEP Thermo Fisher Cat# PG82080 Chloro-acetamide Thermo Fisher Cat# A15238.30 Trypsin/EDTA Gibco Cat# 25300054 Hydroxylamine Thermo Fisher Cat# A15398.0I Acetonitrile Thermo Fisher Cat# 047138.M1 Formic acid Thermo Fisher Cat# 28905 Sorafenib (Bay 43–9006) MedChemExpress Cat# HY-10201 Ulixertinib (BVD-523) MedChemExpress Cat# HY-15816 Zegocractin (CM-4620) MedChemExpress Cat# HY-101942 Critical commercial assays EasySep TM Direct Human PBMC Isolation Kit STEMCELL Technologies Cat# 19654 Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x genomics Cat# PN-1000283 Chromium Fixed RNA Kit, Human Transcriptome 10x genomics Cat# PN-1000476 Dead cell removal kit Miltenyi Biotec Cat# 130-090-101 Quick-DNA/RNA Miniprep Kit Zymo Cat# D7005T Dead cell apoptosis kit, Alexa 488 w/Annexin V PI Invitrogen Cat# V13241 RNeasy® Mini Kit Qiagen Cat# 74104 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Cat# 4368814 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Nextera Library Prep Kit Illumina Cat# FC-131-1096 (Continued on next page) Cell Reports 45, 116903, February 24, 2026 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER MinElute PCR purification Qiagen Cat# 28004 KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 CUTANA TM CUT&RUN Library Prep Kit EpiCypher Cat# 14-1001 Qubit 1X dsDNA HS Assay Kit Thermo Fisher Cat# Q33230 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Duolink® In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Oligonucleotides qPCR primers for IL8, GAPDH, and RN18S1: see Table S2 This study N/A gRNA sequences for STIM1, ORAI1, SPTAN1, and RAF1 knockouts: see Table S3 This study N/A Plasmid lentiCRISPRv2 Addgene Cat# 73179-LV Lenti-iCas9-neo Addgene Cat# 85400 Lenti-multi-Guide plasmid Addgene Cat# 85401 pCDH-CMV-Nluc Addgene Cat#73038 pLenti CMV GFP Puro Addgene Cat# 658-5 pcDNA3-Venus-173-N-ORAI1 Addgene Cat# 87618 pcDNA3.1-STIM1-Venus-173-C Addgene Cat# 87619 pCDH-CMV-Nluc Addgene Cat# 73038 Biological samples Human peripheral blood mononuclear cells (PBMCs) This study N/A THP-1 cells ATCC TIB-202 Lenti-X 293T Takara Bio Cat# 632180 Human CD14 + primary monocytes Bloodworks Bio 4570–61 Antibodies IL8 antibody Invitrogen Cat# 12808842; RRID: AB_2784632 Anti-Ras antibody Abcam Cat# AB108602; RRID: AB_10891004 Raf1 antibody Abcam Cat# AB137435; RRID: AB_3674177 Goat anti-Mouse Alexa Fluor 488 Life Technologies Cat# A32723; RRID: AB_2633275 Goat anti-Rabbit, Alexa Fluor 594 Life Technologies Cat# A32740; RRID: AB_2762824 Anti-Spectrin alpha chain (nonerythroid) Antibody MilliporeSigma Cat# MAB1622; RRID: AB_94295 p-ERK1/2 antibody Cell Signaling Cat# 9101; RRID: AB_331646 ERK1/2 antibody Cell Signaling Cat# 4695; RRID: AB_390779 IRDye 680RD Goat anti-Mouse Li-Cor Cat# 926-68070; RRID: AB_10956588 IRDye 800CW Goat anti-Rabbit Li-Cor Cat# 926-32211; RRID: AB_621843 c-Jun Antibody Thermo Fisher Cat# MA5-15881; RRID: AB_11153582 β-actin Santa Cruz Cat# SC-47778; RRID: AB_626632 Deposited data snRNA-seq/snATAC-seq, ATAC, bulk-RNA, CUT&RUN data This paper NCBI GEO: GSE262146 CRISPRi screen data: See Data S1 This paper NCBI GEO: GSE213184 scRNA-seq data of drug treatment This paper NCBI GEO: GSE291575 Phospho-proteomics: See Data S2 This paper N/A Software and algorithms R 4.2.3 CRAN project https://www.r-project.org/ Seurat R package Version: 4.3.0.1 (Continued on next page) 16 Cell Reports 45, 116903, February 24, 2026 ..

    Software:

    Article Title: A shear stress-responsive pathway in monocytes drives cardiopulmonary bypass-induced inflammation via spectrin/RAF1/store-operated calcium entry.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals and peptides RPMI-1640 Gibco Cat# 11875093 Fetal Bovine Serum, heat inactivated Gibco Cat# 16140-071 Penicillin/Streptomycin Gibco Cat# 15140122 DMEM Gibco Cat# 10566016 PBS Gibco Cat# 10010023 TransIT-Lenti Transfection Reagent Mirus Bio Cat# MIR 6603 FR180204 (ERK Inhibitor II) Cayman Cat# 15544 DMSO Alfa Aesar Cat# 42780 eBioscience TM Protein Transport Inhibitor Cocktail Thermo Fisher Cat# 00-4980-93 IC Fixation Buffer Thermo Fisher Cat# 00-8222-49 Permeabilization Buffer Thermo Fisher Cat# 00-8333-56 eBioscience TM Flow Cytometry Staining Buffer Thermo Fisher Cat# 00-4222-26 Proteinase K Zymo Cat# D3001-2-5 Polybrene MilliporeSigma Cat# TR-1003 Puromycin MedChemExpress Cat# HY-B1743 Doxycycline Thermo Fisher Cat# J60579.22 Propidium iodide MilliporeSigma Cat# P4170 Paraformaldehyde, methanol-free, 16% Electron Microscopy Sciences Cat# 15710 Triton X-100 (DNAse, RNAse, protease free) Acros Organics Cat# 327371000 RIPA lysis buffer Thermo Fisher Cat# 89900 SRC-selective tyrosine kinase inhibitor PP1 MilliporeSigma Cat# 5678091MG Ammonium Bicarbonate Thermo Fisher Cat# 393210010 Urea Thermo Fisher Cat# 29700 TCEP Thermo Fisher Cat# PG82080 Chloro-acetamide Thermo Fisher Cat# A15238.30 Trypsin/EDTA Gibco Cat# 25300054 Hydroxylamine Thermo Fisher Cat# A15398.0I Acetonitrile Thermo Fisher Cat# 047138.M1 Formic acid Thermo Fisher Cat# 28905 Sorafenib (Bay 43–9006) MedChemExpress Cat# HY-10201 Ulixertinib (BVD-523) MedChemExpress Cat# HY-15816 Zegocractin (CM-4620) MedChemExpress Cat# HY-101942 Critical commercial assays EasySep TM Direct Human PBMC Isolation Kit STEMCELL Technologies Cat# 19654 Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x genomics Cat# PN-1000283 Chromium Fixed RNA Kit, Human Transcriptome 10x genomics Cat# PN-1000476 Dead cell removal kit Miltenyi Biotec Cat# 130-090-101 Quick-DNA/RNA Miniprep Kit Zymo Cat# D7005T Dead cell apoptosis kit, Alexa 488 w/Annexin V PI Invitrogen Cat# V13241 RNeasy® Mini Kit Qiagen Cat# 74104 High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Cat# 4368814 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Nextera Library Prep Kit Illumina Cat# FC-131-1096 (Continued on next page) Cell Reports 45, 116903, February 24, 2026 15 .. REAGENT or RESOURCE SOURCE IDENTIFIER MinElute PCR purification Qiagen Cat# 28004 KAPA Library Quantification Kit KAPA Biosystems Cat# KK4824 CUTANA TM CUT&RUN Library Prep Kit EpiCypher Cat# 14-1001 Qubit 1X dsDNA HS Assay Kit Thermo Fisher Cat# Q33230 Pierce TM bicinchoninic acid (BCA) assay kit Thermo Fisher Cat# 23225 Duolink® In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Oligonucleotides qPCR primers for IL8, GAPDH, and RN18S1: see Table S2 This study N/A gRNA sequences for STIM1, ORAI1, SPTAN1, and RAF1 knockouts: see Table S3 This study N/A Plasmid lentiCRISPRv2 Addgene Cat# 73179-LV Lenti-iCas9-neo Addgene Cat# 85400 Lenti-multi-Guide plasmid Addgene Cat# 85401 pCDH-CMV-Nluc Addgene Cat#73038 pLenti CMV GFP Puro Addgene Cat# 658-5 pcDNA3-Venus-173-N-ORAI1 Addgene Cat# 87618 pcDNA3.1-STIM1-Venus-173-C Addgene Cat# 87619 pCDH-CMV-Nluc Addgene Cat# 73038 Biological samples Human peripheral blood mononuclear cells (PBMCs) This study N/A THP-1 cells ATCC TIB-202 Lenti-X 293T Takara Bio Cat# 632180 Human CD14 + primary monocytes Bloodworks Bio 4570–61 Antibodies IL8 antibody Invitrogen Cat# 12808842; RRID: AB_2784632 Anti-Ras antibody Abcam Cat# AB108602; RRID: AB_10891004 Raf1 antibody Abcam Cat# AB137435; RRID: AB_3674177 Goat anti-Mouse Alexa Fluor 488 Life Technologies Cat# A32723; RRID: AB_2633275 Goat anti-Rabbit, Alexa Fluor 594 Life Technologies Cat# A32740; RRID: AB_2762824 Anti-Spectrin alpha chain (nonerythroid) Antibody MilliporeSigma Cat# MAB1622; RRID: AB_94295 p-ERK1/2 antibody Cell Signaling Cat# 9101; RRID: AB_331646 ERK1/2 antibody Cell Signaling Cat# 4695; RRID: AB_390779 IRDye 680RD Goat anti-Mouse Li-Cor Cat# 926-68070; RRID: AB_10956588 IRDye 800CW Goat anti-Rabbit Li-Cor Cat# 926-32211; RRID: AB_621843 c-Jun Antibody Thermo Fisher Cat# MA5-15881; RRID: AB_11153582 β-actin Santa Cruz Cat# SC-47778; RRID: AB_626632 Deposited data snRNA-seq/snATAC-seq, ATAC, bulk-RNA, CUT&RUN data This paper NCBI GEO: GSE262146 CRISPRi screen data: See Data S1 This paper NCBI GEO: GSE213184 scRNA-seq data of drug treatment This paper NCBI GEO: GSE291575 Phospho-proteomics: See Data S2 This paper N/A Software and algorithms R 4.2.3 CRAN project https://www.r-project.org/ Seurat R package Version: 4.3.0.1 (Continued on next page) 16 Cell Reports 45, 116903, February 24, 2026 ..



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    Fig. 1. Generation of Jurkat cell lines stably producing APP. (A) Immunoblot of endogenous APP in Jurkat, 293T/17, and HeLa cells carrying APP-targeting shRNA (APP KD) or control shRNA. (B) qPCR analysis of relative APP mRNA levels in cells presented in (A). Differences from the reference level of 1 set for HeLa control cells were analyzed using one-sample t-test (n ¼ 4 each). Significant differences are marked with asterisks (***p < 0.001). (C) Immunoblot of APP, <t>Orai1,</t> and STIM1 in Jurkat cells stably expressing APP, APPSWE or an empty cassette (control), compared with wildtype HeLa cells. (D) FACS analysis of Y188-labeled Jurkat APP cells and APPSWE cells. Y188-labeled and mock-labeled 293T/17 cells were analyzed in parallel to set a threshold for Y188- positive cells (horizontal line). The x-axis of both histograms shows the Y188 signal normalized to cell size (FL1-H/FSC) on a logarithmic scale.
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    Fig. 1. Generation of Jurkat cell lines stably producing APP. (A) Immunoblot of endogenous APP in Jurkat, 293T/17, and HeLa cells carrying APP-targeting shRNA (APP KD) or control shRNA. (B) qPCR analysis of relative APP mRNA levels in cells presented in (A). Differences from the reference level of 1 set for HeLa control cells were analyzed using one-sample t-test (n ¼ 4 each). Significant differences are marked with asterisks (***p < 0.001). (C) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing APP, APPSWE or an empty cassette (control), compared with wildtype HeLa cells. (D) FACS analysis of Y188-labeled Jurkat APP cells and APPSWE cells. Y188-labeled and mock-labeled 293T/17 cells were analyzed in parallel to set a threshold for Y188- positive cells (horizontal line). The x-axis of both histograms shows the Y188 signal normalized to cell size (FL1-H/FSC) on a logarithmic scale.

    Journal: Biochemical and biophysical research communications

    Article Title: Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

    doi: 10.1016/j.bbrc.2016.08.072

    Figure Lengend Snippet: Fig. 1. Generation of Jurkat cell lines stably producing APP. (A) Immunoblot of endogenous APP in Jurkat, 293T/17, and HeLa cells carrying APP-targeting shRNA (APP KD) or control shRNA. (B) qPCR analysis of relative APP mRNA levels in cells presented in (A). Differences from the reference level of 1 set for HeLa control cells were analyzed using one-sample t-test (n ¼ 4 each). Significant differences are marked with asterisks (***p < 0.001). (C) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing APP, APPSWE or an empty cassette (control), compared with wildtype HeLa cells. (D) FACS analysis of Y188-labeled Jurkat APP cells and APPSWE cells. Y188-labeled and mock-labeled 293T/17 cells were analyzed in parallel to set a threshold for Y188- positive cells (horizontal line). The x-axis of both histograms shows the Y188 signal normalized to cell size (FL1-H/FSC) on a logarithmic scale.

    Article Snippet: Human APP and ORAI1 coding sequences originated from Addgene plasmids #30137, #30145 and #22754, and were cloned MluI-NotI into a modified pLVTH vector (Addgene #12262), in which eGFP and the H1 promoter were replacedwith an IRES-PURO-WPRE cassette.

    Techniques: Stable Transfection, Western Blot, shRNA, Control, Expressing, Labeling

    Fig. 2. Microscopic analysis of SOCE in APP-producing Jurkat cells. (A) SOCE measurements in Fura-4F-loaded Jurkat cells that produced APP (n ¼ 13), APPSWE (n ¼ 12) and control cells with an empty cassette (n ¼ 13). (B) SOCE measurements in Jurkat cells that produced dominant-negative Orai1 (Orai1 DN; n ¼ 5) and control cells (n ¼ 5). Shown are averaged traces from representative experiments and calculated mean values with standard error. Differences from control cells were analyzed using unpaired t-test, and significant dif- ferences are marked with asterisks (***p < 0.001).

    Journal: Biochemical and biophysical research communications

    Article Title: Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

    doi: 10.1016/j.bbrc.2016.08.072

    Figure Lengend Snippet: Fig. 2. Microscopic analysis of SOCE in APP-producing Jurkat cells. (A) SOCE measurements in Fura-4F-loaded Jurkat cells that produced APP (n ¼ 13), APPSWE (n ¼ 12) and control cells with an empty cassette (n ¼ 13). (B) SOCE measurements in Jurkat cells that produced dominant-negative Orai1 (Orai1 DN; n ¼ 5) and control cells (n ¼ 5). Shown are averaged traces from representative experiments and calculated mean values with standard error. Differences from control cells were analyzed using unpaired t-test, and significant dif- ferences are marked with asterisks (***p < 0.001).

    Article Snippet: Human APP and ORAI1 coding sequences originated from Addgene plasmids #30137, #30145 and #22754, and were cloned MluI-NotI into a modified pLVTH vector (Addgene #12262), in which eGFP and the H1 promoter were replacedwith an IRES-PURO-WPRE cassette.

    Techniques: Produced, Control, Dominant Negative Mutation

    Fig. 3. Analysis of SOCE in Jurkat cells producing C99 fragments. (A) Immunoblot of small APP fragments (detected with Y188 antibody) in Jurkat cells stably expressing APPSWE, C99 cDNA, or an empty cassette (control) compared with wildtype HeLa cells. DAPT treatment (5 mM for 5 h) is indicated below the gels. (B) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing C99 cDNA, C99IND cDNA, or an empty cassette (control) compared with wildtype HeLa cells. (C) qPCR analysis of ORAI1 and STIM1 mRNA levels in C99- producing Jurkat cells relative to those in Jurkat control cells. Differences were analyzed using one-sample t-test (n ¼ 3 each). (D) SOCE measurements in Fura-4F-loaded Jurkat C99 cells (n ¼ 11), C99IND cells (n ¼ 11) and control cells (n ¼ 12). Traces are from representative experiments. The data were analyzed using unpaired t-test.

    Journal: Biochemical and biophysical research communications

    Article Title: Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

    doi: 10.1016/j.bbrc.2016.08.072

    Figure Lengend Snippet: Fig. 3. Analysis of SOCE in Jurkat cells producing C99 fragments. (A) Immunoblot of small APP fragments (detected with Y188 antibody) in Jurkat cells stably expressing APPSWE, C99 cDNA, or an empty cassette (control) compared with wildtype HeLa cells. DAPT treatment (5 mM for 5 h) is indicated below the gels. (B) Immunoblot of APP, Orai1, and STIM1 in Jurkat cells stably expressing C99 cDNA, C99IND cDNA, or an empty cassette (control) compared with wildtype HeLa cells. (C) qPCR analysis of ORAI1 and STIM1 mRNA levels in C99- producing Jurkat cells relative to those in Jurkat control cells. Differences were analyzed using one-sample t-test (n ¼ 3 each). (D) SOCE measurements in Fura-4F-loaded Jurkat C99 cells (n ¼ 11), C99IND cells (n ¼ 11) and control cells (n ¼ 12). Traces are from representative experiments. The data were analyzed using unpaired t-test.

    Article Snippet: Human APP and ORAI1 coding sequences originated from Addgene plasmids #30137, #30145 and #22754, and were cloned MluI-NotI into a modified pLVTH vector (Addgene #12262), in which eGFP and the H1 promoter were replacedwith an IRES-PURO-WPRE cassette.

    Techniques: Western Blot, Stable Transfection, Expressing, Control

    Fig. 4. Analysis of SOCE in HeLa cells depleted of APP. (A) SOCE measurements in Fura-2-loaded HeLa cells stably expressing APP-targeting shRNA (APP KD; n ¼ 8), ORAI1-targeting shRNA (ORAI1 KD; n ¼ 3) and control shRNA (control; n ¼ 8). The stores were depleted with 20 mM CPA in Ca2þ-free buffer containing 0.5 mM EGTA for 400 s, and SOCE was initiated by the re-addition of 2 mM Ca2þ. Shown are averaged (black) and individual (gray) SOCE traces from representative experiments. The data were analyzed using unpaired t- test, and significant differences from controls are marked with asterisks (***p < 0.001). (B) qPCR analysis of ORAI1 and STIM1 mRNAs in cells presented in (A). Differences from the reference level of 1 set for controls were analyzed using one-sample t-test (n ¼ 3 each). Statistical significance is marked with asterisks (***p < 0.001).

    Journal: Biochemical and biophysical research communications

    Article Title: Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein.

    doi: 10.1016/j.bbrc.2016.08.072

    Figure Lengend Snippet: Fig. 4. Analysis of SOCE in HeLa cells depleted of APP. (A) SOCE measurements in Fura-2-loaded HeLa cells stably expressing APP-targeting shRNA (APP KD; n ¼ 8), ORAI1-targeting shRNA (ORAI1 KD; n ¼ 3) and control shRNA (control; n ¼ 8). The stores were depleted with 20 mM CPA in Ca2þ-free buffer containing 0.5 mM EGTA for 400 s, and SOCE was initiated by the re-addition of 2 mM Ca2þ. Shown are averaged (black) and individual (gray) SOCE traces from representative experiments. The data were analyzed using unpaired t- test, and significant differences from controls are marked with asterisks (***p < 0.001). (B) qPCR analysis of ORAI1 and STIM1 mRNAs in cells presented in (A). Differences from the reference level of 1 set for controls were analyzed using one-sample t-test (n ¼ 3 each). Statistical significance is marked with asterisks (***p < 0.001).

    Article Snippet: Human APP and ORAI1 coding sequences originated from Addgene plasmids #30137, #30145 and #22754, and were cloned MluI-NotI into a modified pLVTH vector (Addgene #12262), in which eGFP and the H1 promoter were replacedwith an IRES-PURO-WPRE cassette.

    Techniques: Stable Transfection, Expressing, shRNA, Control

    Fig. 1. POST binds Orai1 and is expressed in the ER and plasma membrane. (A) HA epitope-tagged POST specifically binds myc-tagged Orai1 coexpressed in HEK 293 cells. Cell lysates were immunoprecipitated with HA-agarose and stained on Western blot (WB) with myc- or HA-antibody conjugated to HRP. (B) Endogenous POST binds Orai1. Jurkat cell lysates were immunoprecipitated with Orai1, POST antibody, or preimmune rabbit IgG, and probed on WB with the indicated antibody (TrueBlot). Lysates of HEK 293 expressing myc-Orai1 (labeled as myc-Or1) and HA-POST were used as markers. Note that store depletion (cells treated with 1 μM thapsigargin for 10 min in Ca2+-free Ringer’s solution) did not affect POST-Orai1 interaction. (C) Live confocal fluorescence images of HEK 293 cells coexpressing POST-GFP and Cherry-STIM1. POST was localized in the cell periphery and intracellular membrane network, where it precisely colocalized with Cherry STIM1 (ER). Note the fraction of POST that is expressed in the cell periphery, where STIM1 is not expressed.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: POST, partner of stromal interaction molecule 1 (STIM1), targets STIM1 to multiple transporters.

    doi: 10.1073/pnas.1117231108

    Figure Lengend Snippet: Fig. 1. POST binds Orai1 and is expressed in the ER and plasma membrane. (A) HA epitope-tagged POST specifically binds myc-tagged Orai1 coexpressed in HEK 293 cells. Cell lysates were immunoprecipitated with HA-agarose and stained on Western blot (WB) with myc- or HA-antibody conjugated to HRP. (B) Endogenous POST binds Orai1. Jurkat cell lysates were immunoprecipitated with Orai1, POST antibody, or preimmune rabbit IgG, and probed on WB with the indicated antibody (TrueBlot). Lysates of HEK 293 expressing myc-Orai1 (labeled as myc-Or1) and HA-POST were used as markers. Note that store depletion (cells treated with 1 μM thapsigargin for 10 min in Ca2+-free Ringer’s solution) did not affect POST-Orai1 interaction. (C) Live confocal fluorescence images of HEK 293 cells coexpressing POST-GFP and Cherry-STIM1. POST was localized in the cell periphery and intracellular membrane network, where it precisely colocalized with Cherry STIM1 (ER). Note the fraction of POST that is expressed in the cell periphery, where STIM1 is not expressed.

    Article Snippet: In-frame subcloning of the human Orai1 coding sequence (NM_032790; Origene TC124465) into the ATC-TAP vector generated the N-terminal TAP-Orai1 cDNA.

    Techniques: Clinical Proteomics, Membrane, Immunoprecipitation, Staining, Western Blot, Expressing, Labeling