Transfection:Article Title: Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches
Article Snippet: .. HEK293T cells (ATCC CRL‐3216) Complete growth medium for HEK293 cells (see recipe) with and without P/S Opti‐MEM reduced‐serum medium (Gibco, cat. no. 31985‐062) TransIT‐2020 transfection reagent (Mirus, cat. no. MIR5400) Plasmids: pCDH‐Str‐KDEL (ER lumen‐facing hook; Addgene, cat. no.65307) pCDH‐Str‐Ii (ER cytosolic‐facing hook; Addgene, cat. no. 65313) psPAX lentiviral packaging plasmid (Addgene, cat. no. 12260) pMD2.G VSV‐G envelope plasmid (Addgene, cat. no. 12259) Dulbecco's phosphate‐buffered saline (DPBS) without Ca 2+ /Mg 2+ (Sigma‐Aldrich, cat. no. D8537) SH‐SY5Y cells stably expressing SBP‐HiBiT‐Tau, SBP‐HiBit‐TNFα, or SBP‐HiBit‐IL1β (see Support Protocol ) Complete growth medium for SH‐SY5Y cells (see recipe) with and without P/S Polybrene (Millipore, cat. no. TR‐1003‐G) Puromycin (Sigma‐Aldrich, cat. no. P9620) Anti‐streptavidin antibody (Santa Cruz Biotechnology, cat. no. sc‐52236) for validation of hook construct Biotin (Sigma‐Aldrich, cat. no. B4639) 10‐cm tissue culture dishes (Corning, cat. no. 353003) 37°C, 5% CO 2 incubator 0.45‐μm low protein‐binding filter (VWR, cat. no. 28145‐479) .. 89 ml DMEM (Sigma‐Aldrich, cat. no. D6429) 10 ml heat‐inactivated fetal bovine serum (FBS, Gibco, cat. no.10270‐106) 1 ml penicillin‐streptomycin (P/S; Gibco, cat. no. 15140‐122) Mix thoroughly Store up to 2 weeks at 4°C Warm to 37°C before use
Article Title: A Sensitive and Versatile Cell‐Based Assay Combines Luminescence and Trapping Approaches to Monitor Unconventional Protein Secretion
Article Snippet: .. At 50% confluence, HEK293T cells grown in complete medium without penicillin/streptomycin were transiently transfected with a lentiviral vector, either pCDH‐Str‐KDEL (Addgene plasmid #65307) for Strep‐KDEL protein expression with Strep directed to the ER lumen, or pCDH_Str‐Ii (Addgene plasmid # 65313) for Strep‐Ii protein expression with Strep directed to the cytosol, together with the HIV packaging plasmid psPAX2 and the plasmid pMD2G, which codes for the vesicular stomatitis virus envelope glycoprotein G. After transfection (24 h), the medium was refreshed, and after an additional 24 h, virus was collected and filtered through a 0.45 μm low protein binding membrane (VWR International, #28145‐479), and concentrated on sucrose by ultracentrifugation at 17 000 g for 1.5 h, at 4°C. ..
Plasmid Preparation:Article Title: Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches
Article Snippet: .. HEK293T cells (ATCC CRL‐3216) Complete growth medium for HEK293 cells (see recipe) with and without P/S Opti‐MEM reduced‐serum medium (Gibco, cat. no. 31985‐062) TransIT‐2020 transfection reagent (Mirus, cat. no. MIR5400) Plasmids: pCDH‐Str‐KDEL (ER lumen‐facing hook; Addgene, cat. no.65307) pCDH‐Str‐Ii (ER cytosolic‐facing hook; Addgene, cat. no. 65313) psPAX lentiviral packaging plasmid (Addgene, cat. no. 12260) pMD2.G VSV‐G envelope plasmid (Addgene, cat. no. 12259) Dulbecco's phosphate‐buffered saline (DPBS) without Ca 2+ /Mg 2+ (Sigma‐Aldrich, cat. no. D8537) SH‐SY5Y cells stably expressing SBP‐HiBiT‐Tau, SBP‐HiBit‐TNFα, or SBP‐HiBit‐IL1β (see Support Protocol ) Complete growth medium for SH‐SY5Y cells (see recipe) with and without P/S Polybrene (Millipore, cat. no. TR‐1003‐G) Puromycin (Sigma‐Aldrich, cat. no. P9620) Anti‐streptavidin antibody (Santa Cruz Biotechnology, cat. no. sc‐52236) for validation of hook construct Biotin (Sigma‐Aldrich, cat. no. B4639) 10‐cm tissue culture dishes (Corning, cat. no. 353003) 37°C, 5% CO 2 incubator 0.45‐μm low protein‐binding filter (VWR, cat. no. 28145‐479) .. 89 ml DMEM (Sigma‐Aldrich, cat. no. D6429) 10 ml heat‐inactivated fetal bovine serum (FBS, Gibco, cat. no.10270‐106) 1 ml penicillin‐streptomycin (P/S; Gibco, cat. no. 15140‐122) Mix thoroughly Store up to 2 weeks at 4°C Warm to 37°C before use
Article Title: A Sensitive and Versatile Cell‐Based Assay Combines Luminescence and Trapping Approaches to Monitor Unconventional Protein Secretion
Article Snippet: .. At 50% confluence, HEK293T cells grown in complete medium without penicillin/streptomycin were transiently transfected with a lentiviral vector, either pCDH‐Str‐KDEL (Addgene plasmid #65307) for Strep‐KDEL protein expression with Strep directed to the ER lumen, or pCDH_Str‐Ii (Addgene plasmid # 65313) for Strep‐Ii protein expression with Strep directed to the cytosol, together with the HIV packaging plasmid psPAX2 and the plasmid pMD2G, which codes for the vesicular stomatitis virus envelope glycoprotein G. After transfection (24 h), the medium was refreshed, and after an additional 24 h, virus was collected and filtered through a 0.45 μm low protein binding membrane (VWR International, #28145‐479), and concentrated on sucrose by ultracentrifugation at 17 000 g for 1.5 h, at 4°C. ..
Saline:Article Title: Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches
Article Snippet: .. HEK293T cells (ATCC CRL‐3216) Complete growth medium for HEK293 cells (see recipe) with and without P/S Opti‐MEM reduced‐serum medium (Gibco, cat. no. 31985‐062) TransIT‐2020 transfection reagent (Mirus, cat. no. MIR5400) Plasmids: pCDH‐Str‐KDEL (ER lumen‐facing hook; Addgene, cat. no.65307) pCDH‐Str‐Ii (ER cytosolic‐facing hook; Addgene, cat. no. 65313) psPAX lentiviral packaging plasmid (Addgene, cat. no. 12260) pMD2.G VSV‐G envelope plasmid (Addgene, cat. no. 12259) Dulbecco's phosphate‐buffered saline (DPBS) without Ca 2+ /Mg 2+ (Sigma‐Aldrich, cat. no. D8537) SH‐SY5Y cells stably expressing SBP‐HiBiT‐Tau, SBP‐HiBit‐TNFα, or SBP‐HiBit‐IL1β (see Support Protocol ) Complete growth medium for SH‐SY5Y cells (see recipe) with and without P/S Polybrene (Millipore, cat. no. TR‐1003‐G) Puromycin (Sigma‐Aldrich, cat. no. P9620) Anti‐streptavidin antibody (Santa Cruz Biotechnology, cat. no. sc‐52236) for validation of hook construct Biotin (Sigma‐Aldrich, cat. no. B4639) 10‐cm tissue culture dishes (Corning, cat. no. 353003) 37°C, 5% CO 2 incubator 0.45‐μm low protein‐binding filter (VWR, cat. no. 28145‐479) .. 89 ml DMEM (Sigma‐Aldrich, cat. no. D6429) 10 ml heat‐inactivated fetal bovine serum (FBS, Gibco, cat. no.10270‐106) 1 ml penicillin‐streptomycin (P/S; Gibco, cat. no. 15140‐122) Mix thoroughly Store up to 2 weeks at 4°C Warm to 37°C before use
Stable Transfection:Article Title: Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches
Article Snippet: .. HEK293T cells (ATCC CRL‐3216) Complete growth medium for HEK293 cells (see recipe) with and without P/S Opti‐MEM reduced‐serum medium (Gibco, cat. no. 31985‐062) TransIT‐2020 transfection reagent (Mirus, cat. no. MIR5400) Plasmids: pCDH‐Str‐KDEL (ER lumen‐facing hook; Addgene, cat. no.65307) pCDH‐Str‐Ii (ER cytosolic‐facing hook; Addgene, cat. no. 65313) psPAX lentiviral packaging plasmid (Addgene, cat. no. 12260) pMD2.G VSV‐G envelope plasmid (Addgene, cat. no. 12259) Dulbecco's phosphate‐buffered saline (DPBS) without Ca 2+ /Mg 2+ (Sigma‐Aldrich, cat. no. D8537) SH‐SY5Y cells stably expressing SBP‐HiBiT‐Tau, SBP‐HiBit‐TNFα, or SBP‐HiBit‐IL1β (see Support Protocol ) Complete growth medium for SH‐SY5Y cells (see recipe) with and without P/S Polybrene (Millipore, cat. no. TR‐1003‐G) Puromycin (Sigma‐Aldrich, cat. no. P9620) Anti‐streptavidin antibody (Santa Cruz Biotechnology, cat. no. sc‐52236) for validation of hook construct Biotin (Sigma‐Aldrich, cat. no. B4639) 10‐cm tissue culture dishes (Corning, cat. no. 353003) 37°C, 5% CO 2 incubator 0.45‐μm low protein‐binding filter (VWR, cat. no. 28145‐479) .. 89 ml DMEM (Sigma‐Aldrich, cat. no. D6429) 10 ml heat‐inactivated fetal bovine serum (FBS, Gibco, cat. no.10270‐106) 1 ml penicillin‐streptomycin (P/S; Gibco, cat. no. 15140‐122) Mix thoroughly Store up to 2 weeks at 4°C Warm to 37°C before use
Expressing:Article Title: Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches
Article Snippet: .. HEK293T cells (ATCC CRL‐3216) Complete growth medium for HEK293 cells (see recipe) with and without P/S Opti‐MEM reduced‐serum medium (Gibco, cat. no. 31985‐062) TransIT‐2020 transfection reagent (Mirus, cat. no. MIR5400) Plasmids: pCDH‐Str‐KDEL (ER lumen‐facing hook; Addgene, cat. no.65307) pCDH‐Str‐Ii (ER cytosolic‐facing hook; Addgene, cat. no. 65313) psPAX lentiviral packaging plasmid (Addgene, cat. no. 12260) pMD2.G VSV‐G envelope plasmid (Addgene, cat. no. 12259) Dulbecco's phosphate‐buffered saline (DPBS) without Ca 2+ /Mg 2+ (Sigma‐Aldrich, cat. no. D8537) SH‐SY5Y cells stably expressing SBP‐HiBiT‐Tau, SBP‐HiBit‐TNFα, or SBP‐HiBit‐IL1β (see Support Protocol ) Complete growth medium for SH‐SY5Y cells (see recipe) with and without P/S Polybrene (Millipore, cat. no. TR‐1003‐G) Puromycin (Sigma‐Aldrich, cat. no. P9620) Anti‐streptavidin antibody (Santa Cruz Biotechnology, cat. no. sc‐52236) for validation of hook construct Biotin (Sigma‐Aldrich, cat. no. B4639) 10‐cm tissue culture dishes (Corning, cat. no. 353003) 37°C, 5% CO 2 incubator 0.45‐μm low protein‐binding filter (VWR, cat. no. 28145‐479) .. 89 ml DMEM (Sigma‐Aldrich, cat. no. D6429) 10 ml heat‐inactivated fetal bovine serum (FBS, Gibco, cat. no.10270‐106) 1 ml penicillin‐streptomycin (P/S; Gibco, cat. no. 15140‐122) Mix thoroughly Store up to 2 weeks at 4°C Warm to 37°C before use
Article Title: A Sensitive and Versatile Cell‐Based Assay Combines Luminescence and Trapping Approaches to Monitor Unconventional Protein Secretion
Article Snippet: .. At 50% confluence, HEK293T cells grown in complete medium without penicillin/streptomycin were transiently transfected with a lentiviral vector, either pCDH‐Str‐KDEL (Addgene plasmid #65307) for Strep‐KDEL protein expression with Strep directed to the ER lumen, or pCDH_Str‐Ii (Addgene plasmid # 65313) for Strep‐Ii protein expression with Strep directed to the cytosol, together with the HIV packaging plasmid psPAX2 and the plasmid pMD2G, which codes for the vesicular stomatitis virus envelope glycoprotein G. After transfection (24 h), the medium was refreshed, and after an additional 24 h, virus was collected and filtered through a 0.45 μm low protein binding membrane (VWR International, #28145‐479), and concentrated on sucrose by ultracentrifugation at 17 000 g for 1.5 h, at 4°C. ..
Biomarker Discovery:Article Title: Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches
Article Snippet: .. HEK293T cells (ATCC CRL‐3216) Complete growth medium for HEK293 cells (see recipe) with and without P/S Opti‐MEM reduced‐serum medium (Gibco, cat. no. 31985‐062) TransIT‐2020 transfection reagent (Mirus, cat. no. MIR5400) Plasmids: pCDH‐Str‐KDEL (ER lumen‐facing hook; Addgene, cat. no.65307) pCDH‐Str‐Ii (ER cytosolic‐facing hook; Addgene, cat. no. 65313) psPAX lentiviral packaging plasmid (Addgene, cat. no. 12260) pMD2.G VSV‐G envelope plasmid (Addgene, cat. no. 12259) Dulbecco's phosphate‐buffered saline (DPBS) without Ca 2+ /Mg 2+ (Sigma‐Aldrich, cat. no. D8537) SH‐SY5Y cells stably expressing SBP‐HiBiT‐Tau, SBP‐HiBit‐TNFα, or SBP‐HiBit‐IL1β (see Support Protocol ) Complete growth medium for SH‐SY5Y cells (see recipe) with and without P/S Polybrene (Millipore, cat. no. TR‐1003‐G) Puromycin (Sigma‐Aldrich, cat. no. P9620) Anti‐streptavidin antibody (Santa Cruz Biotechnology, cat. no. sc‐52236) for validation of hook construct Biotin (Sigma‐Aldrich, cat. no. B4639) 10‐cm tissue culture dishes (Corning, cat. no. 353003) 37°C, 5% CO 2 incubator 0.45‐μm low protein‐binding filter (VWR, cat. no. 28145‐479) .. 89 ml DMEM (Sigma‐Aldrich, cat. no. D6429) 10 ml heat‐inactivated fetal bovine serum (FBS, Gibco, cat. no.10270‐106) 1 ml penicillin‐streptomycin (P/S; Gibco, cat. no. 15140‐122) Mix thoroughly Store up to 2 weeks at 4°C Warm to 37°C before use
Construct:Article Title: Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches
Article Snippet: .. HEK293T cells (ATCC CRL‐3216) Complete growth medium for HEK293 cells (see recipe) with and without P/S Opti‐MEM reduced‐serum medium (Gibco, cat. no. 31985‐062) TransIT‐2020 transfection reagent (Mirus, cat. no. MIR5400) Plasmids: pCDH‐Str‐KDEL (ER lumen‐facing hook; Addgene, cat. no.65307) pCDH‐Str‐Ii (ER cytosolic‐facing hook; Addgene, cat. no. 65313) psPAX lentiviral packaging plasmid (Addgene, cat. no. 12260) pMD2.G VSV‐G envelope plasmid (Addgene, cat. no. 12259) Dulbecco's phosphate‐buffered saline (DPBS) without Ca 2+ /Mg 2+ (Sigma‐Aldrich, cat. no. D8537) SH‐SY5Y cells stably expressing SBP‐HiBiT‐Tau, SBP‐HiBit‐TNFα, or SBP‐HiBit‐IL1β (see Support Protocol ) Complete growth medium for SH‐SY5Y cells (see recipe) with and without P/S Polybrene (Millipore, cat. no. TR‐1003‐G) Puromycin (Sigma‐Aldrich, cat. no. P9620) Anti‐streptavidin antibody (Santa Cruz Biotechnology, cat. no. sc‐52236) for validation of hook construct Biotin (Sigma‐Aldrich, cat. no. B4639) 10‐cm tissue culture dishes (Corning, cat. no. 353003) 37°C, 5% CO 2 incubator 0.45‐μm low protein‐binding filter (VWR, cat. no. 28145‐479) .. 89 ml DMEM (Sigma‐Aldrich, cat. no. D6429) 10 ml heat‐inactivated fetal bovine serum (FBS, Gibco, cat. no.10270‐106) 1 ml penicillin‐streptomycin (P/S; Gibco, cat. no. 15140‐122) Mix thoroughly Store up to 2 weeks at 4°C Warm to 37°C before use
Low Protein Binding:Article Title: Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches
Article Snippet: .. HEK293T cells (ATCC CRL‐3216) Complete growth medium for HEK293 cells (see recipe) with and without P/S Opti‐MEM reduced‐serum medium (Gibco, cat. no. 31985‐062) TransIT‐2020 transfection reagent (Mirus, cat. no. MIR5400) Plasmids: pCDH‐Str‐KDEL (ER lumen‐facing hook; Addgene, cat. no.65307) pCDH‐Str‐Ii (ER cytosolic‐facing hook; Addgene, cat. no. 65313) psPAX lentiviral packaging plasmid (Addgene, cat. no. 12260) pMD2.G VSV‐G envelope plasmid (Addgene, cat. no. 12259) Dulbecco's phosphate‐buffered saline (DPBS) without Ca 2+ /Mg 2+ (Sigma‐Aldrich, cat. no. D8537) SH‐SY5Y cells stably expressing SBP‐HiBiT‐Tau, SBP‐HiBit‐TNFα, or SBP‐HiBit‐IL1β (see Support Protocol ) Complete growth medium for SH‐SY5Y cells (see recipe) with and without P/S Polybrene (Millipore, cat. no. TR‐1003‐G) Puromycin (Sigma‐Aldrich, cat. no. P9620) Anti‐streptavidin antibody (Santa Cruz Biotechnology, cat. no. sc‐52236) for validation of hook construct Biotin (Sigma‐Aldrich, cat. no. B4639) 10‐cm tissue culture dishes (Corning, cat. no. 353003) 37°C, 5% CO 2 incubator 0.45‐μm low protein‐binding filter (VWR, cat. no. 28145‐479) .. 89 ml DMEM (Sigma‐Aldrich, cat. no. D6429) 10 ml heat‐inactivated fetal bovine serum (FBS, Gibco, cat. no.10270‐106) 1 ml penicillin‐streptomycin (P/S; Gibco, cat. no. 15140‐122) Mix thoroughly Store up to 2 weeks at 4°C Warm to 37°C before use
Article Title: A Sensitive and Versatile Cell‐Based Assay Combines Luminescence and Trapping Approaches to Monitor Unconventional Protein Secretion
Article Snippet: .. At 50% confluence, HEK293T cells grown in complete medium without penicillin/streptomycin were transiently transfected with a lentiviral vector, either pCDH‐Str‐KDEL (Addgene plasmid #65307) for Strep‐KDEL protein expression with Strep directed to the ER lumen, or pCDH_Str‐Ii (Addgene plasmid # 65313) for Strep‐Ii protein expression with Strep directed to the cytosol, together with the HIV packaging plasmid psPAX2 and the plasmid pMD2G, which codes for the vesicular stomatitis virus envelope glycoprotein G. After transfection (24 h), the medium was refreshed, and after an additional 24 h, virus was collected and filtered through a 0.45 μm low protein binding membrane (VWR International, #28145‐479), and concentrated on sucrose by ultracentrifugation at 17 000 g for 1.5 h, at 4°C. ..
Virus:Article Title: A Sensitive and Versatile Cell‐Based Assay Combines Luminescence and Trapping Approaches to Monitor Unconventional Protein Secretion
Article Snippet: .. At 50% confluence, HEK293T cells grown in complete medium without penicillin/streptomycin were transiently transfected with a lentiviral vector, either pCDH‐Str‐KDEL (Addgene plasmid #65307) for Strep‐KDEL protein expression with Strep directed to the ER lumen, or pCDH_Str‐Ii (Addgene plasmid # 65313) for Strep‐Ii protein expression with Strep directed to the cytosol, together with the HIV packaging plasmid psPAX2 and the plasmid pMD2G, which codes for the vesicular stomatitis virus envelope glycoprotein G. After transfection (24 h), the medium was refreshed, and after an additional 24 h, virus was collected and filtered through a 0.45 μm low protein binding membrane (VWR International, #28145‐479), and concentrated on sucrose by ultracentrifugation at 17 000 g for 1.5 h, at 4°C. ..
Membrane:Article Title: A Sensitive and Versatile Cell‐Based Assay Combines Luminescence and Trapping Approaches to Monitor Unconventional Protein Secretion
Article Snippet: .. At 50% confluence, HEK293T cells grown in complete medium without penicillin/streptomycin were transiently transfected with a lentiviral vector, either pCDH‐Str‐KDEL (Addgene plasmid #65307) for Strep‐KDEL protein expression with Strep directed to the ER lumen, or pCDH_Str‐Ii (Addgene plasmid # 65313) for Strep‐Ii protein expression with Strep directed to the cytosol, together with the HIV packaging plasmid psPAX2 and the plasmid pMD2G, which codes for the vesicular stomatitis virus envelope glycoprotein G. After transfection (24 h), the medium was refreshed, and after an additional 24 h, virus was collected and filtered through a 0.45 μm low protein binding membrane (VWR International, #28145‐479), and concentrated on sucrose by ultracentrifugation at 17 000 g for 1.5 h, at 4°C. ..
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