apc101  (Alomone Labs)

Journal: Frontiers in Oncology

Article Title: Regulation of Proliferation by a Mitochondrial Potassium Channel in Pancreatic Ductal Adenocarcinoma Cells

doi: 10.3389/fonc.2017.00239

Figure Lengend Snippet: Inhibition of mitochondrial Kv1.3 kills PANC-1 cells. (A) PANC-1 cells were treated or left untreated with different membrane permeant Kv1.3 inhibitors (PAPTP 10 µM, PCARBTP 10 µM, and clofazimine 20 µM) for 24 h. Cell death was determined by staining with an Alexa568 coupled Annexin V by fluorescent microscopy. The images are representative of three different replicates. (B) Quantification of Annexin V-positive cells from the experiments shown in (A) : represented is the percentage of Annexin V-positive cells ± SEM ( n = 3; ** p

Article Snippet: After blocking with a 10% solution of defatted milk, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-Kv1.3 (1:200, rabbit polyclonal; Alomone Labs APC-101); anti-GAPDH (1:1,000, mouse monoclonal; Millipore MAB374).

Techniques: Inhibition, Staining, Microscopy

Journal: Frontiers in Oncology

Article Title: Regulation of Proliferation by a Mitochondrial Potassium Channel in Pancreatic Ductal Adenocarcinoma Cells

doi: 10.3389/fonc.2017.00239

Figure Lengend Snippet: Sublethal inhibition of mitochondrial Kv1.3 leads to cell cycle alterations. (A) FACS analysis of cell cycle of PANC-1 cells untreated or treated with clofazimine 1 µM and ShK 100 nM for 24 h. The distribution was determined by staining cells with 50 µg/mL Propidium Iodide and the acquisition was performed by a FACSantoII (Beckton Dickson). The plots are representative of three separated determinations. Light blue peaks represent apoptotic cells, while red peaks represent cells in G1 and G2/M phases. S phase is represented by the area with blue straight lines. A sub-G1 peak is due to dead cells. In the graph of untreated cells the labels and the arrows identify the different populations that are reported. (B) Cell cycle distribution in PANC-1 cells pretreated (lower panels) or not (upper panels) with 50 µM Mitotempo or 20 mM N-acetylcysteine (NAC) for 1 h, before the addition of mitochondrial Kv1.3 inhibitors PAPTP and PCARBTP (both at 100 nM) for 24 h. The experiment and the analysis have been performed as in (A) . The plots are representative of three separate measurements.

Article Snippet: After blocking with a 10% solution of defatted milk, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-Kv1.3 (1:200, rabbit polyclonal; Alomone Labs APC-101); anti-GAPDH (1:1,000, mouse monoclonal; Millipore MAB374).

Techniques: Inhibition, FACS, Staining

Journal: Frontiers in Oncology

Article Title: Regulation of Proliferation by a Mitochondrial Potassium Channel in Pancreatic Ductal Adenocarcinoma Cells

doi: 10.3389/fonc.2017.00239

Figure Lengend Snippet: Potassium channel Kv1.3 is expressed in PANC-1 cells. (A) Kv1.3 expression was determined by Western Blot in PANC-1 cells. 60 µg of total protein extract were loaded into a SDS gel and blotted onto a polyvinylidene fluoride (PVDF) membrane. Kv1.3 band was evaluated by immunoblotting with a specific antibody. GAPDH was used as loading control. The blot is a representative image of three different observations. (B) Inhibition of mitochondrial Kv1.3 by different concentration of membrane permeant blockers resulted in a reduction of the MTS signal from PANC-1 cells. Values are reported as percentage respect to untreated sample ± SEM. All compounds were added for 24 h. Staurosporine was used as positive control ( n = 3; *** p

Article Snippet: After blocking with a 10% solution of defatted milk, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-Kv1.3 (1:200, rabbit polyclonal; Alomone Labs APC-101); anti-GAPDH (1:1,000, mouse monoclonal; Millipore MAB374).

Techniques: Expressing, Western Blot, SDS-Gel, Inhibition, Concentration Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation *

doi: 10.1074/jbc.M115.678995

Figure Lengend Snippet: Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).

Article Snippet: Non-permeabilized cells were incubated with anti-Kv1.3 or anti Kv1.5 extracellular primary antibodies (APC101 or APC150, Alomone Labs), whereas permeabilized cells were incubated with anti-Kv1.3 COOH (75-009, NeuroMab) or anti-Kv1.5 COOH (APC004, Alomone Labs), all at a final concentration of 1:50.

Techniques: Mutagenesis, Activation Assay, Transfection, Staining, Expressing