anti ent2 primary antibodies  (Alomone Labs)

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Effects of FPMINT analogues (listed in ) on [ 3 H]uridine uptake by ENT1 and ENT2 [ 3 H]uridine uptake (1 μM, 2 μCi/ml) in (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells was measured in the presence of various concentrations of FPMINT analogues listed in (10 nM–100 μM).

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Effects of FPMINT analogues (listed in ) on [ 3 H]uridine uptake by ENT1 and ENT2 [ 3 H]uridine uptake (1 μM, 2 μCi/ml) in (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells was measured in the presence of various concentrations of FPMINT analogues listed in (10 nM–100 μM).

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Competitive inhibition of ENT1 and ENT2 by compound 3c. Kinetic study of [ 3 H]uridine uptake (0.1 μM–1 mM) was measured in (A) PK15NTD/ENT1 and (D) PK15NTD/ENT2 cells in the presence of various concentrations of compound 3c (0, 0.01, 0.1, 1 and 10 µM) (B,E) 1/ V versus 1/[S] plots of each curve in (A) and (D) , respectively (C,F) The plot of slopes of each line in (B) and (E) , respectively, versus the concentration of compound 3c . Data are presented as mean ± SE of three experiments.

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: Inhibition, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Effects of compound 3c on cytotoxicity and protein expressions of ENT1 and ENT2. Cytotoxicity of compound 3c was measured after 24 h or 48 h treatment of PK15NTD/ENT1 and PK15NTD/ENT2 cells with various concentrations of compound 3c (0.5, 5 and 50 μM) or a vehicle (0.5% DMSO, as a control) (A) The cell viability and (B) cell membrane integrity were determined by the MTT assay and LDH release, respectively (C) PK15NTD/ENT1 and (D) PK15NTD/ENT2 cells were incubated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for 24 or 48 h. Western blotting assay was performed to determine the protein expression levels of ENT1 and ENT2, with β-actin as an internal reference. Cell surface proteins of (E) PK15NTD/ENT1 and (F) PK15NTD/ENT2 cells were biotinylated and treated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for 24 or 48 h. After cleavage of extracellular biotin, internalized biotinylated proteins were precipitated with immobilized streptavidin beads and detected with an anti-ENT1 and anti-ENT2 antibodies by western blotting. Total ENT1 and ENT2 from whole-cell lysates were also detected for a comparison. β-actin served as an internal reference. Representative blots are from three independent experiments. Data are presented as mean ± SE of three experiments.

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: MTT Assay, Incubation, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Reversibility of the inhibitory effects of compound 3c on ENT1 and ENT2 (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells were incubated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for different time (0–60 min) The cells were then washed five times. After wash-out for every single time [ 3 H]uridine uptake (1 μM, 2 μCi/ml) was measured. Data are presented as mean ± SE of three experiments. * p < 0.05 compared with the fifth wash.

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: Incubation

Journal: bioRxiv

Article Title: Targeting pathogenic Lafora bodies in Lafora disease using an antibody-enzyme fusion

doi: 10.1101/679407

Figure Lengend Snippet: VAL-0417 uptake and polysaccharide reduction in cell culture. (A) Rat1 cells and HEK293 cells stably expressing PTG and PP1Cα after 20-hour treatment with increasing concentrations of VAL-0417. VAL-0417 levels were assessed by Western blotting using an anti-AMY2A antibody. ENT2 levels were also quantified by Western blotting. (B) Polysaccharide levels in Rat and HEK293-PTG/PP1Cα cells after 20-hour treatment with VAL-0417, expressed as a percent of controls. Data shown are a mean of 4 measurements ± SE. Statistical significance is indicated: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: For Western analyses, 20 μg of total cell lysates were separated on SDS-PAGE and protein transferred to nitrocellulose membranes that were probed with antibodies against human pancreatic α-amylase (Abcam #ab21156) and ENT2 (Alomone Labs #ANT-052) followed by appropriate HRP-conjugated secondary antibodies and chemiluminescence.

Techniques: Cell Culture, Stable Transfection, Expressing, Western Blot