Journal:

Article Title: Functional implications of the localization and activity of acid-sensitive channels in rat peripheral nervous system

doi: 10.1073/pnas.042688199

Figure Lengend Snippet: Characterization of ASIC antibodies. (A) Western blots from HEK cells transfected with ASIC-1, -2a, -3, and -4 cDNAs probed with affinity-purified specific antibodies. (B) Immunocytochemistry of HEK cells transfected with ASIC-1, -2, and -3 cDNAs tagged with FLAG epitope. (Top) Confocal images of cells labeled with the corresponding anti-ASIC antibodies (green); (Middle) labeling with monoclonal anti-FLAG antibody (red); (Bottom) the merge of the previous two images (yellow).

Article Snippet: Primary anti-ASIC antibodies and a commercial antibody that recognizes the N terminus of rat ASIC2 (ASIC2-N) (Alomone Laboratories, Jerusalem) were diluted 1:2,000 to 1:6,000.

Techniques: Western Blot, Transfection, Affinity Purification, Immunocytochemistry, FLAG-tag, Labeling

Journal: Frontiers in Oncology

Article Title: Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer

doi: 10.3389/fonc.2022.904742

Figure Lengend Snippet: ASIC channel activity in A549, WI-38, and HLF cells. (A) Whole-cell configuration of the patch clamp technique and protocol of experiments. Membrane voltage was clamped at –50 mV in all patches. (B) Representative traces showing ASIC1a-like currents activated by a rapid drop of extracellular pH from 7.4 to 5.5, the currents were reversibly inhibited by 10 μM benzamil (derivative of amiloride). (C) Distribution of transient peak amplitudes at pH 5.5 elicited ASIC1a-like currents in A549 cells. (D) The effect of mambalgin-2 on ASIC1a-like currents in A549 cells. (E) ASIC1a-like currents in WI-38 cells. (F) Distribution of transient peak amplitudes in WI-38 cells at pH 5.5 elicited ASIC1a-like currents. (G) The effect of mambalgin-2 on ASIC1a-like currents in WI-38 cells. (H) An absence of acidification-evoked currents in HLF cells.

Article Snippet: The membranes were then incubated overnight at 4°C with primary antibodies against ASIC1a (mouse, 1:1,000, SMC-427, StressMarq Biosciences, Victoria, Canada), α-ENaC (rabbit, 1:1,000, ABIN1841945, Antibodies-Online), or γ-ENaC (mouse, 1:1,000, ABIN1865926, Antibodies-Online), washed three times with TBS + 0.1% Tween-20, and incubated with HRP-conjugated secondary anti-rabbit antibody (1:5,000, 111-035-003, Jackson ImmunoResearch), in the case of α-ENaC or anti-mouse antibody (1:5,000, 715-035-150, Jackson ImmunoResearch) in the cases of ASIC1a or γ-ENaC for 1 h (20°C).

Techniques: Activity Assay, Patch Clamp

Journal: Frontiers in Oncology

Article Title: Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer

doi: 10.3389/fonc.2022.904742

Figure Lengend Snippet: Analysis of mambalgin-2 and amiloride action at ASIC1a and ASIC1a/α-ENaC/γ-ENaC channels in Xenopus laevis oocytes. (A) Two-electrode configuration of the patch-clamp technique and protocol of experiments. Representative current traces recorded in X. laevis oocytes expressing the hASIC1a (B) and hASIC1a/α-ENAC/γ-ENAC (C) channels; traces for non-injected oocytes were used as control and are shown in Figure S9 . The pre-incubation with mambalgin-2 was 15 s (shown off time scale), the stimulation phase (pH 5.0) was 7 s, and the recovery phase is not shown. (D) Dose–response curves for mambalgin-2 inhibitory effect. For each mambalgin-2 concentration, the current response was normalized to the control experiment. Each data point represents an average from independent experiments in different oocytes ± SEM (n =11 for both channels). The fitted curves are described by single-component Hill’s equation. (E) Dose–response curves for amiloride inhibitory effect. The data points are shown as normalized average ± SEM (n = 3 for both channels).

Article Snippet: The membranes were then incubated overnight at 4°C with primary antibodies against ASIC1a (mouse, 1:1,000, SMC-427, StressMarq Biosciences, Victoria, Canada), α-ENaC (rabbit, 1:1,000, ABIN1841945, Antibodies-Online), or γ-ENaC (mouse, 1:1,000, ABIN1865926, Antibodies-Online), washed three times with TBS + 0.1% Tween-20, and incubated with HRP-conjugated secondary anti-rabbit antibody (1:5,000, 111-035-003, Jackson ImmunoResearch), in the case of α-ENaC or anti-mouse antibody (1:5,000, 715-035-150, Jackson ImmunoResearch) in the cases of ASIC1a or γ-ENaC for 1 h (20°C).

Techniques: Patch Clamp, Expressing, Injection, Incubation, Concentration Assay

Journal: Frontiers in Oncology

Article Title: Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer

doi: 10.3389/fonc.2022.904742

Figure Lengend Snippet: Influence of knockdown of ASIC1, α-ENaC, and γ-ENaC expression on mambalgin-2 activity in A549 lung cancer cells. Dose–response effects of mambalgin-2 on viability of A549 cells after cell transfection with scramble siRNA or siRNA specific for ASIC1a (A) , α-ENaC (B) , and γ-ENaC. (C) Data are presented as % of the control (untreated cells) ± SEM (n = 4). The gene knockdown confirmation is shown in Figures S1A–E , and the analysis of dose–response curves is given in Table 1 . (D) Representative pictures of wounds for A549 cells incubated at pH 5.5 in the presence of 1 µM of mambalgin-2 upon knockdown of ASIC1a, α-ENaC, and γ-ENaC genes. Scale bar = 100 µm. Analysis of gene knockdown influence on the migration of A549 cells in the absence of mambalgin-2 is given in Figures S1F, G . (E) Wound area occupied by migrating A549 cells. Data are presented as % of the wound surface, occupied by migrating cells ± SEM (n = 8); ### (p < 0.001) and *** (p < 0.001) indicate significant difference between the data groups by one-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: The membranes were then incubated overnight at 4°C with primary antibodies against ASIC1a (mouse, 1:1,000, SMC-427, StressMarq Biosciences, Victoria, Canada), α-ENaC (rabbit, 1:1,000, ABIN1841945, Antibodies-Online), or γ-ENaC (mouse, 1:1,000, ABIN1865926, Antibodies-Online), washed three times with TBS + 0.1% Tween-20, and incubated with HRP-conjugated secondary anti-rabbit antibody (1:5,000, 111-035-003, Jackson ImmunoResearch), in the case of α-ENaC or anti-mouse antibody (1:5,000, 715-035-150, Jackson ImmunoResearch) in the cases of ASIC1a or γ-ENaC for 1 h (20°C).

Techniques: Expressing, Activity Assay, Transfection, Incubation, Migration

Journal: Frontiers in Oncology

Article Title: Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer

doi: 10.3389/fonc.2022.904742

Figure Lengend Snippet: Parameters describing the effect of ASIC1a, α-ENaC, and γ-ENaC knockdown on the anti-proliferative activity of mambalgin-2 in A549 cells.

Article Snippet: The membranes were then incubated overnight at 4°C with primary antibodies against ASIC1a (mouse, 1:1,000, SMC-427, StressMarq Biosciences, Victoria, Canada), α-ENaC (rabbit, 1:1,000, ABIN1841945, Antibodies-Online), or γ-ENaC (mouse, 1:1,000, ABIN1865926, Antibodies-Online), washed three times with TBS + 0.1% Tween-20, and incubated with HRP-conjugated secondary anti-rabbit antibody (1:5,000, 111-035-003, Jackson ImmunoResearch), in the case of α-ENaC or anti-mouse antibody (1:5,000, 715-035-150, Jackson ImmunoResearch) in the cases of ASIC1a or γ-ENaC for 1 h (20°C).

Techniques: Activity Assay