Journal: PLoS genetics
Article Title: The association of the vanin-1 N131S variant with blood pressure is mediated by endoplasmic reticulum-associated degradation and loss of function.
doi: 10.1371/journal.pgen.1004641
Figure Lengend Snippet: Figure 3. N131S mutation leads to significantly lower total and surface vainin-1 protein and fractional pantetheinase activity compared to WT, whereas T26I does not. HEK293 cells stably expressing WT, T26I or N131S vanin-1 were generated using the G418 selection method. HEK293 cells transfected with empty vector (EV) were used as a negative control. (A) Cells were lysed and 30 mg of total protein were subjected to 8% SDS-PAGE and Western blot analysis using a rabbit polyclonal anti-vanin-1 antibody (Pierce antibodies) (n = 3). b-actin serves as a loading control. (B) Cells were incubated with the membrane-impermeable biotinylation reagent Sulfo-NHS SS-Biotin (Pierce) and biotinylated surface proteins were affinity-purified using immobilized neutravidin-conjugated agarose beads (Pierce). Surface proteins were subjected to 8% SDS- PAGE and Western blot analysis using a rabbit polyclonal anti-vanin-1 antibody (Pierce antibodies) (n = 3). The protein band intensity in (A) and (B) was quantified using ImageJ software from the NIH. Data is reported as mean 6 SEM. ** p,0.01. NS: non-significant. (C) A cell-based fluorescence assay was used to evaluate vanin-1 variants’ pantetheinase activity according to published procedure with modifications [36]. The substrate, pantothenate-7-amino-4-methylcoumarin (Pantothenate-AMC) was chemically synthesized. Cells were lysed and 10 mg of total proteins were added to the substrate Pantothenate-AMC (5 mM) in a 100 mL final volume. Fluorescence signals at excitation 350 nm and emission 460 nm measuring the released AMC were recorded every 3 min using a fluorescence plate reader. A 60-min kinetics assay in four replicates and three biological replicates was carried out. Buffer only and HEK293 cells transfected with empty vector (EV) were used as negative controls for non-specific pantetheinase activity. doi:10.1371/journal.pgen.1004641.g003
Article Snippet: The rabbit polyclonal anti-vanin-1 antibody came from Pierce antibodies, the mouse monoclonal anti-transferrin antibody from Santa Cruz Biotechnology, the mouse monoclonal anti-b-actin antibody from Sigma, and the rabbit polyclonal anti-ubiquitin antibody from Cell Signaling.
Techniques: Mutagenesis, Activity Assay, Stable Transfection, Expressing, Generated, Selection, Transfection, Plasmid Preparation, Negative Control, SDS Page, Western Blot, Control, Incubation, Membrane, Affinity Purification, Software, Fluorescence, Synthesized
![Figure 3. N131S mutation leads to significantly lower total and surface vainin-1 protein and fractional pantetheinase activity compared to WT, whereas T26I does not. HEK293 cells stably expressing WT, T26I or N131S vanin-1 were generated using the G418 selection method. HEK293 cells transfected with empty vector (EV) were used as a negative control. (A) Cells were lysed and 30 mg of total protein were subjected to 8% SDS-PAGE and Western blot analysis using a rabbit <t>polyclonal</t> anti-vanin-1 antibody (Pierce antibodies) (n = 3). b-actin serves as a loading control. (B) Cells were incubated with the membrane-impermeable biotinylation reagent Sulfo-NHS SS-Biotin (Pierce) and biotinylated surface proteins were affinity-purified using immobilized neutravidin-conjugated agarose beads (Pierce). Surface proteins were subjected to 8% SDS- PAGE and Western blot analysis using a rabbit polyclonal anti-vanin-1 antibody (Pierce antibodies) (n = 3). The protein band intensity in (A) and (B) was quantified using ImageJ software from the NIH. Data is reported as mean 6 SEM. ** p,0.01. NS: non-significant. (C) A cell-based fluorescence assay was used to evaluate vanin-1 variants’ pantetheinase activity according to published procedure with modifications [36]. The substrate, pantothenate-7-amino-4-methylcoumarin (Pantothenate-AMC) was chemically synthesized. Cells were lysed and 10 mg of total proteins were added to the substrate Pantothenate-AMC (5 mM) in a 100 mL final volume. Fluorescence signals at excitation 350 nm and emission 460 nm measuring the released AMC were recorded every 3 min using a fluorescence plate reader. A 60-min kinetics assay in four replicates and three biological replicates was carried out. Buffer only and HEK293 cells transfected with empty vector (EV) were used as negative controls for non-specific pantetheinase activity. doi:10.1371/journal.pgen.1004641.g003](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_3454/pm25233454/pm25233454__page6_image1.jpg)
