rabbit polyclonal anti panx1  (Alomone Labs)

Journal: Cell and Tissue Research

Article Title: Nanobody-based Pannexin1 channel inhibitors increase survival after cardiac ischemia/reperfusion

doi: 10.1007/s00441-025-03994-y

Figure Lengend Snippet: PANX1 expression in Dubai camel fibroblasts transfected with mouse Panx1 (DUBCA mPanx1 ). a PANX1 expression and cellular localization were confirmed by immunofluorescence. PANX1 was found in the cell membrane and in the cytosol of DUBCA mPanx1 . The fluorescent signal was almost not detected in parental DUBCA cells and not present in the negative control (omission of primary antibody). DAPI (blue), scale bar = 50 μm. Representative photographs of three independent experiments. b Western blotting reveals bands for PANX1-Gly2 (48 kDa) and for PANX1 -Gly0/Gly1 (41–43 kDa) in DUBCA mPanx1 cells. These bands were virtually absent in parental DUBCA cells. Chinese hamster ovary (CHO) cells transfected with human Panx1-GFP (75 kDa) (CHO hPanx1 ) and empty vector-transfected cells (CHO EV ) were used as a positive and negative control, respectively. β-actin (42 kDa) was used as a loading control. Representative image of two independent experiments

Article Snippet: PANX1 expression was verified by Western blot using a rabbit anti-Panx1 primary antibody (1/1000; Cell Signaling, cat. no. 91137) and the corresponding secondary anti-rabbit antibody (1/5000; Jackson Laboratories).

Techniques: Expressing, Transfection, Immunofluorescence, Membrane, Negative Control, Western Blot, Plasmid Preparation, Control

Journal: Cell and Tissue Research

Article Title: Nanobody-based Pannexin1 channel inhibitors increase survival after cardiac ischemia/reperfusion

doi: 10.1007/s00441-025-03994-y

Figure Lengend Snippet: The specific binding of nanobodies Panx1 channels was verified by flow cytometry analysis. a Flow cytometry analysis shows PANX1 expression in EA.hy926 cells (blue) and its absence in H9c2 cells (orange). The average fluorescence intensity of EA.hy926 cells without primary antibody (red; negative control) was comparable to the H9c2 signal. Representative example of > 3 independent experiments. b , c Immunofluorescence staining confirms the presence of PANX1 (green, left panel) in b (left panel) EA.hy926 cells and b (right panel) absence of immunosignal from the negative control. c PANX1 is not detected in H9c2 cells. Evans blue (red), DAPI (blue), scale bar = 50 μm. Representative example of > 3 independent experiments. d – k EA.hy926 cells were stained with d Nb1, e Nb3, f Nb6, g Nb9, h Nb16, i Nb20, j Nb30, and k the control NbR3b23 at concentrations of 100 nM (blue) or 300 nM (orange). Flow cytometry analysis revealed a low background fluorescent signal in the negative control (red; omission of nanobody). Most nanobodies, except Nb30 and NbR3b23, bound to EA.hy926 cells albeit with different affinities. Representative examples of two independent experiments. l Quantitative RT-PCR analysis confirms the absence of Panx1 mRNA in H9c2 cells. Results are normalized to Panx1 mRNA expression in rat liver (black). Rat kidney samples were used as an additional positive control (grey). N = 3–4. m – s H9c2 were stained with l Nb1, m Nb3, n Nb6, o Nb9, p Nb16, q Nb20, and r Nb30 at a concentration of 300 nM (orange). Flow cytometry analysis revealed a low background fluorescent signal in the negative control (red; omission of nanobody). None of the nanobodies bound to Panx1-deficient H9c2 cells, illustrating the specificity of the nanobodies. Representative examples of two independent experiments

Article Snippet: PANX1 expression was verified by Western blot using a rabbit anti-Panx1 primary antibody (1/1000; Cell Signaling, cat. no. 91137) and the corresponding secondary anti-rabbit antibody (1/5000; Jackson Laboratories).

Techniques: Binding Assay, Flow Cytometry, Expressing, Fluorescence, Negative Control, Immunofluorescence, Staining, Control, Quantitative RT-PCR, Positive Control, Concentration Assay

Journal: Cell and Tissue Research

Article Title: Nanobody-based Pannexin1 channel inhibitors increase survival after cardiac ischemia/reperfusion

doi: 10.1007/s00441-025-03994-y

Figure Lengend Snippet: Nb1 and Nb9 decrease neutrophil adhesion to an activated endothelium. a Quantitative RT-PCR analysis showing the expression levels of Panx1 mRNA in human PLB-985 myeloid leukemia cells upon 3-, 5-, or 7-day DMSO-induced differentiation into neutrophils. EA.hy926 cells were used as a positive control. N = 1. b – d Quantitative RT-PCR analysis demonstrating the expression of icam-1 ( b ), vcam-1 ( c ), and Panx1 ( d ) mRNA in control EA.hy926 cells and after activation with 10 ng/mL hTNFα. N = 3 ** P ≤ 0.01, *** P ≤ 0.001 (two-tailed Student’s t test). e Representative photographs of adhered CFDA-SE-stained PLB-985 cells onto monolayers of activated EA.hy926 cells under control conditions (vehicle) or after incubation of Nb1, Nb9, or NbR3b23 (100 nM). Brightfield photograph (top panels), fluorescence photograph (bottom panels). Scale bar = 100 μm. f – g Quantification of adhered PLB-985 cells onto monolayers of control and activated EA.hy926 cells incubated with vehicle (0.9% NaCl), 2.5 mM probenecid, 100 nM Nb1, Nb9, or NbR3b23. N = 3–4 * P ≤ 0.05, **** P ≤ 0.0001 vs. vehicle (one-way ANOVA)

Article Snippet: PANX1 expression was verified by Western blot using a rabbit anti-Panx1 primary antibody (1/1000; Cell Signaling, cat. no. 91137) and the corresponding secondary anti-rabbit antibody (1/5000; Jackson Laboratories).

Techniques: Quantitative RT-PCR, Expressing, Positive Control, Control, Activation Assay, Two Tailed Test, Staining, Incubation, Fluorescence