Anti-UPF3B/RENT3B Antibody Picoband®

Western blot analysis of UPF3B/RENT3B using anti-UPF3B/RENT3B antibody (PB9843).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Hela whole cell lysates,
Lane 2: human 22RV1 whole cell lysates,
Lane 3: human U87 whole cell lysates,
Lane 4: human Jurkat whole cell lysates,
Lane 5: rat brain tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-UPF3B/RENT3B antigen affinity purified polyclonal antibody (Catalog # PB9843) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for UPF3B/RENT3B at approximately 58 kDa. The expected band size for UPF3B/RENT3B is at 58 kDa.

Cytokine-induced suppression of NMD activity is associated with UPF3B downregulation and attenuated by UPF3 overexpression in β cells. EndoC-βH3 cells were cotransfected with empty vector (E.V.), UPF3A, UPF3B, and/or UPF3BΔ42 (dominant negative of UPF3B) plasmids, and then with Renilla -HBB(PTC − ) and or Renilla -HBB(PTC + ), along with the Firefly plasmid and exposed to cytokine combination (Cyt; 3 ng/mL IL-1β + 10 ng/mL IFN-γ + 10 ng/mL TNF-α) for 18 (h) (A (i, ii) ) mRNA level of Upf3A and Upf3B genes in EndoC-βH3 cells was quantified by RT-qPCR and normalised to tubulin mRNAs. (B) Luciferase activity was measured in the lysate of the transfected cells and represented as NMD activity calculated by dividing luciferase activity of HBB(PTC − )/HBB(PTC + ) as explained in the Methods. The overexpression of UPF3A and UPF3B proteins was examined by Western blot analysis [ Supplementary Figure 4B (i)]. The data are means ± SEM of N = 6. The symbol “ * ” indicates the Bonferroni-corrected paired t -test values of treated versus untreated E.V. (Unt) (A, B) or, otherwise, cytokine (Cyt)-treated E.V. that is designated by a line on top of the bars (B) : * ≤ 0.05; ** ≤ 0.01. ns, nonsignificant.
Index in PubMed under a CC BY license. PMID: 38586449

UPF3 overexpression deteriorates cell viability and reduces insulin content but not secretion in EndoC-βH3 cells. EndoC-βH3 cells were cotransfected with empty vector (E.V.), UPF3A, and/or UPF3B plasmids and exposed to cytokine combination (Cyt; 3 ng/mL IL-1β + 10 ng/mL IFN-γ + 10 ng/mL TNF-α) for 3 days. (A) Cell viability was measured by Alamarblue (i) and caspase-3 activity (ii) assays ( N = 6). (B) Glucose-stimulated insulin secretion (GSIS) (i) and insulin contents (iii) were investigated in the transfected EndoC-βH3 cells. Insulin concentration (ng/mL) was measured by insulin ultrasensitive assay ( N = 6). GSIS index (ii) was calculated by dividing the insulin concentration measured in the treatments at 17 mM by 2 mM of glucose. The data are means ± SEM of N = 6. The symbol “ * ” indicates the Bonferroni-corrected paired t -test values of treated versus untreated E.V. (Unt) or, otherwise, cytokine (Cyt)-treated E.V. that is designated by a line on top of the bars (A) or the Bonferroni-corrected paired t -test values of the corresponding low versus high glucose, that is otherwise designated by lines on top of the bars (B (i) ) : * ≤ 0.05; ** ≤ 0.01; **** ≤ 0.0001. ns, nonsignificant.
Index in PubMed under a CC BY license. PMID: 38586449

IHC analysis of UPF3B/RENT3B using anti-UPF3B/RENT3B antibody (PB9843).
UPF3B/RENT3B was detected in a paraffin-embedded section of mouse testis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 μg/ml rabbit anti-UPF3B/RENT3B Antibody (PB9843) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

IHC analysis of UPF3B/RENT3B using anti-UPF3B/RENT3B antibody (PB9843).
UPF3B/RENT3B was detected in a paraffin-embedded section of rat testis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 μg/ml rabbit anti-UPF3B/RENT3B Antibody (PB9843) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

IHC analysis of UPF3B/RENT3B using anti-UPF3B/RENT3B antibody (PB9843).
UPF3B/RENT3B was detected in a paraffin-embedded section of human placenta tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 μg/ml rabbit anti-UPF3B/RENT3B Antibody (PB9843) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.