rabbit anti hv1  (Alomone Labs)

Journal: Journal of Neuroinflammation

Article Title: Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury

doi: 10.1186/s12974-020-01942-x

Figure Lengend Snippet: Genotype identification in Hv1 −/− mice. a Tail DNA analyzed by PCR. b Hv1 protein deficiency confirmed by Western blotting. c Expression of Hv1 in microglia assessed by double-immunofluorescence labeling with Hv1 (green) and the microglial marker, IBA-1 (red; scale bar, 50 μm). Hv1 was co-localized with the microglial marker, IBA-1, in wild-type (WT) mice. In contrast, Hv1 immunofluorescent signal was rarely observed in Hv1 −/− (KO) mice

Article Snippet: The following primary antibodies were used: rabbit anti-GSDMD (1:1000, Cell Signaling Technology), mouse anti-Caspase-1 (1:500, AdipoGen), mouse anti-NLRP3 (1:1000, AdipoGen), rabbit anti-ASC (1:1000, Cell Signaling Technology), rabbit anti-IL-18 (1:1000, Abclonal), rabbit anti-Hv1 (1:1000, Sigma), rabbit anti-NOS2 (1:1000; Abclonal), mouse anti-NF-L and anti-NF-H (1:1000; Cell Signaling Technology), rabbit anti-TUJ1 (1:1000, Abcam), mouse anti-MAG (1:1000, Santa Cruz), rat anti-MBP (1:1000, Millipore), rabbit anti-β-actin (1:1000, Servicebio), and rabbit anti-GAPDH (1:1000, Servicebio).

Techniques: Western Blot, Expressing, Immunofluorescence, Labeling, Marker

Journal: Journal of Neuroinflammation

Article Title: Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury

doi: 10.1186/s12974-020-01942-x

Figure Lengend Snippet: Spatial and temporal distribution of neuronal pyroptosis and apoptosis after SCI and reduced incidence in Hv1 KO mice. a Representative images of sections showing the pyroptotic marker GSDMD (red), TUNEL (green), and DAPI (blue; scale bar, 500 μm). a The location of the injury core after SCI; b, the area surrounding the injury core after SCI, with apoptosis mainly being located closer to the injury core than pyroptosis (scale bar, 50 μm). b Representative confocal images showing GSDMD (green) and NeuN (red) in sham-operated mice and at 1, 3, and 7 days following SCI (scale bar, 50 μm). c Quantification of the fraction (%) of GSDMD-positive neurons (GSDMD + NeuN + cells/NeuN + cells × 100). d Representative images of neurons showing NeuN (red) and TUNEL (green) in sham-operated mice and at 1, 3, and 7 days following SCI (scale bar, 50 μm). e Quantification of the fraction (%) of TUNEL-positive neurons (TUNEL + NeuN + cells/ NeuN + cells × 100). f Western blotting showing cleaved GSDMD. g Quantification of cleaved-GSDMD protein normalized to β-actin from Western blotting ( n = 5 for each treatment; # p < 0.05 ## p < 0.01, SCI vs. sham treatment; * p < 0.05 ** p < 0.01, KO SCI vs. WT SCI)

Article Snippet: The following primary antibodies were used: rabbit anti-GSDMD (1:1000, Cell Signaling Technology), mouse anti-Caspase-1 (1:500, AdipoGen), mouse anti-NLRP3 (1:1000, AdipoGen), rabbit anti-ASC (1:1000, Cell Signaling Technology), rabbit anti-IL-18 (1:1000, Abclonal), rabbit anti-Hv1 (1:1000, Sigma), rabbit anti-NOS2 (1:1000; Abclonal), mouse anti-NF-L and anti-NF-H (1:1000; Cell Signaling Technology), rabbit anti-TUJ1 (1:1000, Abcam), mouse anti-MAG (1:1000, Santa Cruz), rat anti-MBP (1:1000, Millipore), rabbit anti-β-actin (1:1000, Servicebio), and rabbit anti-GAPDH (1:1000, Servicebio).

Techniques: Marker, TUNEL Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury

doi: 10.1186/s12974-020-01942-x

Figure Lengend Snippet: Hv1 deficiency reduces activation of the NLRP3 inflammasome after SCI. a – c Representative confocal images showing the NLRP3 inflammasome markers (green) NLRP3 ( a ), Caspase-1 ( b ), and ASC ( c ), as well as the neuronal marker, NeuN (red; scale bar, 50 μm). d – f Quantification of the fraction (%) of d NLRP3-positive neurons (NLRP3 + NeuN + cells/NeuN + cells × 100), ( e ) Caspase-1-positive neurons (Caspase-1 + NeuN + cells/NeuN + cells × 100) and f ASC-positive neurons (ASC + NeuN + cells/NeuN + cells × 100). g Western blotting showing NLRP3, caspase-1 p20, ASC, and IL-18 protein levels. h – k Quantification of Western-blot results for NLRP3 (H), caspase-1 p20 ( i ), ASC ( j ), and IL-18 ( k ) normalized to β-actin. l The expression of IL-18 mRNA via real-time PCR. Data are represented as the mean ± SEM ( n = 5 for each treatment; # p < 0.05 ## p < 0.01, SCI vs. sham treatment; * p < 0.05 ** p < 0.01, KO SCI vs. WT SCI)

Article Snippet: The following primary antibodies were used: rabbit anti-GSDMD (1:1000, Cell Signaling Technology), mouse anti-Caspase-1 (1:500, AdipoGen), mouse anti-NLRP3 (1:1000, AdipoGen), rabbit anti-ASC (1:1000, Cell Signaling Technology), rabbit anti-IL-18 (1:1000, Abclonal), rabbit anti-Hv1 (1:1000, Sigma), rabbit anti-NOS2 (1:1000; Abclonal), mouse anti-NF-L and anti-NF-H (1:1000; Cell Signaling Technology), rabbit anti-TUJ1 (1:1000, Abcam), mouse anti-MAG (1:1000, Santa Cruz), rat anti-MBP (1:1000, Millipore), rabbit anti-β-actin (1:1000, Servicebio), and rabbit anti-GAPDH (1:1000, Servicebio).

Techniques: Activation Assay, Marker, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury

doi: 10.1186/s12974-020-01942-x

Figure Lengend Snippet: Hv1 deficiency suppresses microglial ROS generation after SCI. a Representative confocal images of coronal sections showing IBA-1 (red) and 8 hydroxyguanosine (8-OHG) (green) at different time points post-SCI (scale bar, 50 μm). b Quantification of the fraction (%) of 8-OHG and IBA-1 double-positive cells (8-OHG + IBA-1 + cells/IBA-1 + cells × 100) in sham-operated mice and at 1, 3, and 7 days following SCI. c Quantitative analysis of relative ROS levels (DCFH-DA fluorescent intensity normalized to that in sham mice). d Western blotting showing NOS2 and β-actin. e Quantification of NOS2 normalized to β-actin (from Western blotting). Data are represented as the mean ± SEM ( n = 5 for each treatment; ## p < 0.01, SCI vs. sham treatment; * p < 0.05 ** p < 0.01, KO SCI vs. WT SCI)

Article Snippet: The following primary antibodies were used: rabbit anti-GSDMD (1:1000, Cell Signaling Technology), mouse anti-Caspase-1 (1:500, AdipoGen), mouse anti-NLRP3 (1:1000, AdipoGen), rabbit anti-ASC (1:1000, Cell Signaling Technology), rabbit anti-IL-18 (1:1000, Abclonal), rabbit anti-Hv1 (1:1000, Sigma), rabbit anti-NOS2 (1:1000; Abclonal), mouse anti-NF-L and anti-NF-H (1:1000; Cell Signaling Technology), rabbit anti-TUJ1 (1:1000, Abcam), mouse anti-MAG (1:1000, Santa Cruz), rat anti-MBP (1:1000, Millipore), rabbit anti-β-actin (1:1000, Servicebio), and rabbit anti-GAPDH (1:1000, Servicebio).

Techniques: Western Blot

Journal: Journal of Neuroinflammation

Article Title: Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury

doi: 10.1186/s12974-020-01942-x

Figure Lengend Snippet: Hv1 deficiency promotes axonal regeneration and recovery of motor function. a Representative image of Luxol fast-blue staining on days 14 and 28 after SCI (scale bar, 200 μm). b Representative image of GFAP staining (red) and BDA-labeled axons (green) in sham-operated mice and at 28 days after SCI in WT and KO mice (scale bar, 200 μm). c Western blotting showing NF-H, NF-L, TUJ1, MAG, and MBP levels in sham-operated mice and at 14 and 28 days after SCI. d – h Quantification of Western-blot results for NF-H ( d ), NF-L ( e ), TUJ1 ( f ), MAG ( g ), and MBP ( h ) normalized to GAPDH. Values are represented as the mean ± SEM ( n = 5 for each treatment). i Analysis of Basso Mouse Scale (BMS) scores before and after SCI in WT and KO mice ( n = 8 for each genotype; * p < 0.05 ** p < 0.01, KO SCI vs. WT SCI)

Article Snippet: The following primary antibodies were used: rabbit anti-GSDMD (1:1000, Cell Signaling Technology), mouse anti-Caspase-1 (1:500, AdipoGen), mouse anti-NLRP3 (1:1000, AdipoGen), rabbit anti-ASC (1:1000, Cell Signaling Technology), rabbit anti-IL-18 (1:1000, Abclonal), rabbit anti-Hv1 (1:1000, Sigma), rabbit anti-NOS2 (1:1000; Abclonal), mouse anti-NF-L and anti-NF-H (1:1000; Cell Signaling Technology), rabbit anti-TUJ1 (1:1000, Abcam), mouse anti-MAG (1:1000, Santa Cruz), rat anti-MBP (1:1000, Millipore), rabbit anti-β-actin (1:1000, Servicebio), and rabbit anti-GAPDH (1:1000, Servicebio).

Techniques: Staining, Labeling, Western Blot

Journal: Redox Biology

Article Title: Proton channel blockers inhibit Duox activity independent of Hv1 effects

doi: 10.1016/j.redox.2019.101346

Figure Lengend Snippet: High concentrations of Hv1 channel blockers inhibit Duox H 2 O 2 synthesis . H 2 O 2 synthesis by fully differentiated NHBE cells was assayed in the absence and presence of either Zn 2+ (panel a, Tris-Ringers solution) or ClGBI (panels b–d, PBS). Rates of AR oxidation were normalized to assays in the absence of inhibitors but with vehicle. ATP (100 μM) was used to stimulate Ca 2+ dependent Duox activity. All values are means ± s.e.m. Panel a , Zn 2+ inhibited ATP stimulated Duox H 2 O 2 synthesis (IC50 = 0.68 mM, 3–5 lung donors, triplicate cultures each donor). Panels b and c , ClGBI inhibited both baseline Duox activity (IC50 = 0.070 mM, n = 3–6 lung donors, triplicate cultures each donor) and stimulated activity (IC50 = 0.12 mM, n = 3–6 lung donors, triplicate cultures each donor). Panel d , Inhibition of stimulated Duox activity by ClGBI (0.3 mM) was not rescued by addition of superoxide dismutase (20 Units/ml), n = 6 cultures, 3 lung donors.

Article Snippet: Panel f, SDS extract (20 μg) of NHBE (lane 1), Jurkat cells (lane 2) and HEK293T cells (lane 3) were applied to a 10% polyacrylamide SDS gel, transferred and probed with rabbit anti-human Hv1 C-terminal peptide (0.6 μg/ml, ARP35377_P050, Aviva Systems Biology, San Diego CA).

Techniques: Activity Assay, Inhibition

Journal: Redox Biology

Article Title: Proton channel blockers inhibit Duox activity independent of Hv1 effects

doi: 10.1016/j.redox.2019.101346

Figure Lengend Snippet: H + /K + ATPase plays a role in controlling NHBE pHi . Panel a , qPCR determination of mRNA levels in freshly isolated NHBE cells showed ATP12A expression greatly exceeded HVCN1 and was equivalent to Duox1 within the limits of efficiency for different TaqMan® kits and RNA preparations. Transcripts values are relative to GAPDH and are means ± S.E.M, n = 18 lung donors for ATP12A and HVCN1, n = 6 lung donors for Duox1 and Duox2. Panel b , Airway surface liquid pH of NHBE cultures was measured using BCECF in DPBS (pH 7.1). Fluorescence signal was confirmed to be extracellular by removal and replacement of apical solutions. Rapid acidification of control culture mucosal buffer (closed circles) was blocked by addition of Ouabain (1 mM) (squares), mean ± S.E.M. n = 3 cultures, 1 lung donor. Panel c, NHBE cultures were loaded with BCECF-AM and then treated with either vehicle, Ouabain (1 mM), Zn 2+ (100 μM) or both inhibitors, all in DPBS. Only Ouabain containing treatments showed a significant reduction in pHi compared to control, mean ± S.E.M., n = 3. one lung donor, p < 0.05, Tukey-Kramer HSD. Panel d , ATP12A expression in NHBE cells was reduced by shRNA expressing lentivirus (see Supplemental methods). Transcripts relative to β 2 -microglobulin were reduced >90% compared to vector controls, n = 5 cultures, 2 lung donors. Panel e , NHBE cultures with reduced ATP12A expression were loaded with BCECF-AM and pHi was measured. Cells with reduced ATP12A expression had lower pHi, mean ± S.EM, n = 5 cultures, 2 lung donors, p < 0.05, Wilcoxon test. Panel f, SDS extract (20 μg) of NHBE (lane 1), Jurkat cells (lane 2) and HEK293T cells (lane 3) were applied to a 10% polyacrylamide SDS gel, transferred and probed with rabbit anti-human Hv1 C-terminal peptide (0.6 μg/ml, ARP35377_P050, Aviva Systems Biology, San Diego CA). A single band with a Mapp of ~37 kDa was visible in lane 1. This band was absent in HEK293 cells (lane 3) and in a duplicate blot using antibody preincubated with blocking peptide (not shown).

Article Snippet: Panel f, SDS extract (20 μg) of NHBE (lane 1), Jurkat cells (lane 2) and HEK293T cells (lane 3) were applied to a 10% polyacrylamide SDS gel, transferred and probed with rabbit anti-human Hv1 C-terminal peptide (0.6 μg/ml, ARP35377_P050, Aviva Systems Biology, San Diego CA).

Techniques: Isolation, Expressing, Fluorescence, Control, shRNA, Plasmid Preparation, SDS-Gel, Blocking Assay