Incubation:Article Title: Secretogranin II-Derived Peptide Secretoneurin Differentially Stimulates Luteinizing Hormone Secretion by Age and Sex in Ex Vivo Murine Pituitaries†.
Article Snippet: Secretoneurin (SN) is a small, bioactive proteolytic product of secretogranin II (SCG2), a chromogranin protein involved in secretory vesicle packaging.. SCG2 co-aggregates with luteinizing hormone (LH) in pituitary gonadotrope secretory granules and thus has long been D ow naded rom http/academ ic.p.com /biolreprod/advance/10.1093/biolre/ioag058/8512077 by BD -FLC H -U SP user on 15 M arch 2026 UN CO RR EC TE D MA NU SC RI PT 4 associated with GnRH-regulated LH secretion.. Previously, the SCG2-derived peptide SN has been identified as a regulator of LH production and secretion in fish and a murine gonadotropelike cell line (LβT2).
Article Title: Type VIIb secretion system recruits the dedicated cell wall hydrolase EssH to enable effector secretion by Staphylococcus aureus .
Article Snippet: .. For western blot, proteins were transferred to polyvinylidene difluoride membrane, blocked for 1 h with 5% dry milk in PBS-T (phosphate-buffered saline with 0.1% Tween 20) containing 80 μg/mL of human IgG (Sigma-Aldrich) to block protein A, and incubated with primary polyclonal antibodies at a dilution of 1:5,000 for 1 h. Membranes were washed four times for 10 min in PBS-T, incubated with 1:10,000 horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology) for 1 h and then washed four times again for 10 min in PBS-T. Immunoreactive products were revealed by chemiluminescent detection using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). .. The blots were developed on Amersham Hyperfilm ECL (GE Healthcare Life Sciences) or using ChemiDoc imaging system (Bio-Rad).
Article Title: Integrative biology shows DPP4 affects inflammatory response to eclampsia and cell model growth via p65/NLRP3/ASC/Caspase-1 pathway
Article Snippet: Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE (Bio-Rad Laboratories, Catalog No. 4561033, United States) and transferred to PVDF membranes (Millipore, Catalog No. IPVH00010, United States) using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, United States). .. Membranes were blocked with 5% non-fat milk (Thermo Fisher Scientific, United States) in TBST for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies: DPP4 (Abcam, Catalog No. ab133605, UK), IL6 (Cell Signaling Technology, Catalog No. 12153, United States), TNF-α (Cell Signaling Technology, Catalog No. 3707, USA), GAPDH (Cell Signaling Technology, Catalog No. 2118, United States), Caspase-1 (Cell Signaling Technology, Catalog No. 2267, United States), p-Caspase-1 (p17) (Cell Signaling Technology, Catalog No. 3866, United States), ASC (Santa Cruz Biotechnology, Catalog No. sc-514414, United States), p-ASC (PhosphoSolutions, Catalog No. 352-P-ASC, United States), NLRP3 (Abcam, Catalog No. ab214185, UK), p-NLRP3 (Cell Signaling Technology, Catalog No. 12291, United States), p65 (Cell Signaling Technology, Catalog No. 8242, United States), and p-p65 (Cell Signaling Technology, Catalog No. 3033, United States). .. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Catalog No. 7076, United States) for 1 h at room temperature.
Immunoprecipitation:Article Title: HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36
Article Snippet: Soluble proteins were quantified with a Bicinchoninic acid (BCA) Assay (Thermo Fisher), and equal quantities of protein were used for immunoprecipitation. .. Immunoprecipitation was performed by incubating 300–500 μg of protein lysate with 1:100 of primary antibody for 3 h at 4°C, followed by capture of antigen-antibody complexes with 20 μL of protein A or G magnetic beads (Cell Signaling). .. Soluble proteins were quantified with a Bicinchoninic acid (BCA) Assay (Thermo Fisher), and equal quantities of protein were used for immunoprecipitation.
Magnetic Beads:Article Title: HCMV promotes viral reactivation through the coordinated regulation of Notch signaling by UL8 and miR-UL36
Article Snippet: Soluble proteins were quantified with a Bicinchoninic acid (BCA) Assay (Thermo Fisher), and equal quantities of protein were used for immunoprecipitation. .. Immunoprecipitation was performed by incubating 300–500 μg of protein lysate with 1:100 of primary antibody for 3 h at 4°C, followed by capture of antigen-antibody complexes with 20 μL of protein A or G magnetic beads (Cell Signaling). .. Soluble proteins were quantified with a Bicinchoninic acid (BCA) Assay (Thermo Fisher), and equal quantities of protein were used for immunoprecipitation.
Western Blot:Article Title: Type VIIb secretion system recruits the dedicated cell wall hydrolase EssH to enable effector secretion by Staphylococcus aureus .
Article Snippet: .. For western blot, proteins were transferred to polyvinylidene difluoride membrane, blocked for 1 h with 5% dry milk in PBS-T (phosphate-buffered saline with 0.1% Tween 20) containing 80 μg/mL of human IgG (Sigma-Aldrich) to block protein A, and incubated with primary polyclonal antibodies at a dilution of 1:5,000 for 1 h. Membranes were washed four times for 10 min in PBS-T, incubated with 1:10,000 horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology) for 1 h and then washed four times again for 10 min in PBS-T. Immunoreactive products were revealed by chemiluminescent detection using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). .. The blots were developed on Amersham Hyperfilm ECL (GE Healthcare Life Sciences) or using ChemiDoc imaging system (Bio-Rad).
Membrane:Article Title: Type VIIb secretion system recruits the dedicated cell wall hydrolase EssH to enable effector secretion by Staphylococcus aureus .
Article Snippet: .. For western blot, proteins were transferred to polyvinylidene difluoride membrane, blocked for 1 h with 5% dry milk in PBS-T (phosphate-buffered saline with 0.1% Tween 20) containing 80 μg/mL of human IgG (Sigma-Aldrich) to block protein A, and incubated with primary polyclonal antibodies at a dilution of 1:5,000 for 1 h. Membranes were washed four times for 10 min in PBS-T, incubated with 1:10,000 horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology) for 1 h and then washed four times again for 10 min in PBS-T. Immunoreactive products were revealed by chemiluminescent detection using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). .. The blots were developed on Amersham Hyperfilm ECL (GE Healthcare Life Sciences) or using ChemiDoc imaging system (Bio-Rad).
Saline:Article Title: Type VIIb secretion system recruits the dedicated cell wall hydrolase EssH to enable effector secretion by Staphylococcus aureus .
Article Snippet: .. For western blot, proteins were transferred to polyvinylidene difluoride membrane, blocked for 1 h with 5% dry milk in PBS-T (phosphate-buffered saline with 0.1% Tween 20) containing 80 μg/mL of human IgG (Sigma-Aldrich) to block protein A, and incubated with primary polyclonal antibodies at a dilution of 1:5,000 for 1 h. Membranes were washed four times for 10 min in PBS-T, incubated with 1:10,000 horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology) for 1 h and then washed four times again for 10 min in PBS-T. Immunoreactive products were revealed by chemiluminescent detection using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). .. The blots were developed on Amersham Hyperfilm ECL (GE Healthcare Life Sciences) or using ChemiDoc imaging system (Bio-Rad).
Blocking Assay:Article Title: Type VIIb secretion system recruits the dedicated cell wall hydrolase EssH to enable effector secretion by Staphylococcus aureus .
Article Snippet: .. For western blot, proteins were transferred to polyvinylidene difluoride membrane, blocked for 1 h with 5% dry milk in PBS-T (phosphate-buffered saline with 0.1% Tween 20) containing 80 μg/mL of human IgG (Sigma-Aldrich) to block protein A, and incubated with primary polyclonal antibodies at a dilution of 1:5,000 for 1 h. Membranes were washed four times for 10 min in PBS-T, incubated with 1:10,000 horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology) for 1 h and then washed four times again for 10 min in PBS-T. Immunoreactive products were revealed by chemiluminescent detection using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). .. The blots were developed on Amersham Hyperfilm ECL (GE Healthcare Life Sciences) or using ChemiDoc imaging system (Bio-Rad).
Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).
Phospho-proteomics:
Staining:Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).
Purification:Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).
Affinity Chromatography:Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).
Cell Culture:Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis
Article Snippet: .. After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ). .. Cells were washed and incubated with secondary antibody (diluted in 1 % BSA in PBS) for 1 h at room temperature: goat anti-chicken IgY Alexa Fluor 488-conjugate (1:1000; Invitrogen cat. A-11039), donkey anti-human IgG Dylight 550-conjugate (1:1000; Invitrogen cat. SA5-10127), goat anti-mouse IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11029), sheep anti-mouse IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2181), donkey anti-mouse IgG Alexa 555 (1:500; Invitrogen cat. A-31570), goat anti-mouse IgG2a Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-21131) rabbit anti-mouse IgG3 DyLight 549-conjugate (1:5000; Rockland cat. 610-442-043), goat anti-mouse IgM Alexa Fluor 568-conjugate (1:500; Invitrogen cat. A-21043), goat anti-rabbit IgG Alexa Fluor 488-conjugate (1:500; Invitrogen cat. A-11008), sheep anti-rabbit IgG Cy3-conjugate (1:1000; Sigma-Aldrich cat. C2306).
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