primary antibodies against kv1 3  (Alomone Labs)

Journal: Cerebral Cortex (New York, NY)

Article Title: Modulation of pacemaker channel function in a model of thalamocortical hyperexcitability by demyelination and cytokines

doi: 10.1093/cercor/bhab491

Figure Lengend Snippet: Alteration in HCN channel expression in thalamus following general demyelination. ( A , B ) Immunofluorescence staining of the VB complex (horizontal thalamic sections, 40 μm) comparing the expression of HCN4 (A, in red, rb-anti-HCN4, 1:200, Alomone) and HCN2 (B, in red, rb-anti-HCN4, 1:200, Alomone) channels between control C3H/HeJ and Day1. The purified ms-anti- neurofilament antibody (SMI312, pan axonal, 1:200, BioLegend) was used as an axonal marker (SMI312, in green). Cell nuclei were stained with DAPI (in blue). ( C , D ) Bar graphs comparing the intensity of the fluorescence signal (using integrated fluorescence intensity values) for SMI312 (C and D upper traces) and HCN4 and HCN2 (lower traces) between the control C3H/HeJ and Day1. ( E ) Immunofluorescence staining of VB in control C3H/HeJ and Day1 with antibodies against TRIP8b (ms-anti-(constant) TRIP8b, 1:50, NeuroMab, in green) and phosphorylated TRIP8b (rb-α-pS237 antibody, 1:100, YenZym). ( F ) Representative bar graph comparing the intensity of the fluorescence signal for total TRIP8b and pS237 between the two groups indicating a significant reduction for phosphorylated TRIP8b, pS237, on Day1. Scale bars indicate 100 μm. VPL, VPM, and TRN stand for ventral-posterior medial, ventral-posterior lateral, and thalamic reticular nucleus, respectively.

Article Snippet: Sections were washed three times for 10 min in PBS and incubated for 2 h in blocking solution (10% normal goat serum, 3% bovine serum albumin (BSA), 0.3% Triton-X100 in PBS) followed by 48 h of incubation at 4 °C with the following primary antibodies: polyclonal rabbit (rb)-anti-HCN2 (1:200), rb-anti-HCN4 (1:200; Alomone Labs), rb-anti-NeuN (neuronal specific marker, 1:1000; Abcam), mouse purified (ms)-anti-neurofilament marker (pan axonal, cocktail, SMI312; 1:200, BioLegend) and ms-anti-Parvalbumin (PV235, 1:500; Swant).

Techniques: Expressing, Immunofluorescence, Staining, Purification, Marker, Fluorescence

Journal: Journal of Healthcare Engineering

Article Title: Effect of Riluzole on the Expression of HCN2 in Dorsal Root Ganglion Neurons of Diabetic Neuropathic Pain Rats

doi: 10.1155/2022/8313415

Figure Lengend Snippet: Changes of HCN2 level in DRG detected by immunofluorescence analysis. (a) Representative images of the immunofluorescent analysis showing HCN2 (green) expression of L3-5 DRG from control, DNP, DMSO, and riluzole. Scale bar, 100 μ m. (b) Quantification of HCN2-positive neurons in the four different groups. ∗ ( p )

Article Snippet: Following sequential incubation overnight at 4°C with rabbit anti-HCN2 (1 : 200, Alomone Labs) and mouse anti-β -actin (1 : 1000) primary antibody, the blots were rinsed thrice in washing buffer for 5 min each and incubated for 2 h with AP-conjugated goat antirabbit IgG (1 : 1000, Beyotime Biotechnology, Nantong, China) or goat antimouse secondary antibody IgG (1 : 1000).

Techniques: Immunofluorescence, Expressing

Journal: Journal of Healthcare Engineering

Article Title: Effect of Riluzole on the Expression of HCN2 in Dorsal Root Ganglion Neurons of Diabetic Neuropathic Pain Rats

doi: 10.1155/2022/8313415

Figure Lengend Snippet: Changes of HCN2 level in DRG detected by western blotting. (a) Western blotting analysis of HCN2 from L3-5 DRG neurons derived of rats in group control, DNP, DMSO, and riluzole. (b) Quantitative analysis showed that riluzole significantly decreased the expression of HCN2 protein in diabetic rats compared to group DMSO. ∗ ( p )

Article Snippet: Following sequential incubation overnight at 4°C with rabbit anti-HCN2 (1 : 200, Alomone Labs) and mouse anti-β -actin (1 : 1000) primary antibody, the blots were rinsed thrice in washing buffer for 5 min each and incubated for 2 h with AP-conjugated goat antirabbit IgG (1 : 1000, Beyotime Biotechnology, Nantong, China) or goat antimouse secondary antibody IgG (1 : 1000).

Techniques: Western Blot, Derivative Assay, Expressing

Journal: Frontiers in Physiology

Article Title: TRPC6 mediates high glucose-induced mitochondrial fission through activation of CDK5 in cultured human podocytes

doi: 10.3389/fphys.2022.984760

Figure Lengend Snippet: Effects of TRPC6 inhibition on mitochondrial functions in podocytes under HG conditions. (A) JC-1 staining and fluorescence analysis in the control, HG, HG + scramble, HG + siTRPC6, HG + DMSO, and HG + 2-APB groups. Scale bars, 20 μm ( n = 3 independent experiments). (B) MitoSOX Red staining and fluorescence analysis in the six groups. Scale bars, 50 μm ( n = 3 independent experiments). (C) TUNEL staining and quantitation analysis of apoptotic cell number (%) in the six groups. Scale bars, 100 μm ( n = 3 independent experiments). *** p

Article Snippet: Western blotting was performed as described previously ( ) and probed with the following primary antibodies: TRPC6 polyclonal rabbit antibodies (1:200, Alomone Labs, Israel), calpain-1 monoclonal rabbit antibodies (1:1,000, Abcam, United States), Drp1 monoclonal rabbit antibodies, p-Drp1 polyclonal rabbit antibodies, Mfn2 monoclonal rabbit antibodies, Opa1 monoclonal rabbit antibodies (1:1,000, Cell Signaling Technology, United States), or β-Actin rabbit antibody (1:5,000, ABclonal, China).

Techniques: Inhibition, Staining, Fluorescence, TUNEL Assay, Quantitation Assay

Journal: Frontiers in Physiology

Article Title: TRPC6 mediates high glucose-induced mitochondrial fission through activation of CDK5 in cultured human podocytes

doi: 10.3389/fphys.2022.984760

Figure Lengend Snippet: Effects of TRPC6 inhibition on mitochondrial morphology in podocytes under HG conditions. (A) MitoTracker Red and DAPI staining, and analysis of average length and aspect ratio by ImageJ software in the control, HG, HG + scramble, HG + siTRPC6, HG + DMSO and HG + 2-APB groups. Scale bars, 10 μm ( n = 5 independent experiments). (B) Representative electron micrographs in the six groups. Scale bars, 500 nm. (C) Representative Western blot and quantitation analysis of p-Drp1 (Ser616) and total Drp1 in the six groups ( n = 3 independent experiments). (D) Representative Western blot and quantitation analysis of Mfn2 and Opa1 in the six groups ( n = 3 independent experiments). ** p

Article Snippet: Western blotting was performed as described previously ( ) and probed with the following primary antibodies: TRPC6 polyclonal rabbit antibodies (1:200, Alomone Labs, Israel), calpain-1 monoclonal rabbit antibodies (1:1,000, Abcam, United States), Drp1 monoclonal rabbit antibodies, p-Drp1 polyclonal rabbit antibodies, Mfn2 monoclonal rabbit antibodies, Opa1 monoclonal rabbit antibodies (1:1,000, Cell Signaling Technology, United States), or β-Actin rabbit antibody (1:5,000, ABclonal, China).

Techniques: Inhibition, Staining, Software, Western Blot, Quantitation Assay

Journal: Frontiers in Physiology

Article Title: TRPC6 mediates high glucose-induced mitochondrial fission through activation of CDK5 in cultured human podocytes

doi: 10.3389/fphys.2022.984760

Figure Lengend Snippet: TRPC6 mediates HG-induced mitochondrial fission through calpain-1 activation in podocytes. (A) Calpain activity assay data in the control, HG, HG + scramble, HG + siTRPC6, HG + DMSO, and HG + 2-APB groups ( n = 5 independent experiments). (B) MitoTracker Red and DAPI staining, and analysis of average length and aspect ratio by ImageJ software in the control, HG, HG + scramble, HG + siCalpain-1, HG + DMSO, and HG + calpeptin groups. Scale bars, 10 μm ( n = 5 independent experiments). (C) Representative Western blot and quantitation analysis of p-Drp1 (Ser616) and total Drp1 in the six groups ( n = 3 independent experiments). (D) Representative Western blot and quantitation analysis of Mfn2 and Opa1 in the six groups ( n = 3 independent experiments). * p

Article Snippet: Western blotting was performed as described previously ( ) and probed with the following primary antibodies: TRPC6 polyclonal rabbit antibodies (1:200, Alomone Labs, Israel), calpain-1 monoclonal rabbit antibodies (1:1,000, Abcam, United States), Drp1 monoclonal rabbit antibodies, p-Drp1 polyclonal rabbit antibodies, Mfn2 monoclonal rabbit antibodies, Opa1 monoclonal rabbit antibodies (1:1,000, Cell Signaling Technology, United States), or β-Actin rabbit antibody (1:5,000, ABclonal, China).

Techniques: Activation Assay, Activity Assay, Staining, Software, Western Blot, Quantitation Assay

Journal: Frontiers in Physiology

Article Title: TRPC6 mediates high glucose-induced mitochondrial fission through activation of CDK5 in cultured human podocytes

doi: 10.3389/fphys.2022.984760

Figure Lengend Snippet: TRPC6 activates CDK5 through the Ca 2+ /calpain-1 pathway which contributes to HG-induced mitochondrial fission in podocytes. (A) CDK5 kinase activity assay data in the control, HG, HG + scramble, HG + siCalpain-1, HG + DMSO and HG + Calpeptin groups (left), and in the control, HG, HG + scramble, HG + siTRPC6, HG + DMSO and HG + 2-APB groups (right) ( n = 5 independent experiments). (B) MitoTracker Red and DAPI staining, and analysis of average length and aspect ratio by ImageJ software in the control, HG, HG + scramble, HG + siCDK5, HG + DMSO, and HG + roscovitine groups. Scale bars, 10 μm ( n = 5 independent experiments). (C) Representative Western blot and quantitation analysis of p-Drp1 (Ser616) and total Drp1 in the six groups ( n = 3 independent experiments). (D) Representative Western blot and quantitation analysis of Mfn2 and Opa1 in the six groups ( n = 3 independent experiments). ** p

Article Snippet: Western blotting was performed as described previously ( ) and probed with the following primary antibodies: TRPC6 polyclonal rabbit antibodies (1:200, Alomone Labs, Israel), calpain-1 monoclonal rabbit antibodies (1:1,000, Abcam, United States), Drp1 monoclonal rabbit antibodies, p-Drp1 polyclonal rabbit antibodies, Mfn2 monoclonal rabbit antibodies, Opa1 monoclonal rabbit antibodies (1:1,000, Cell Signaling Technology, United States), or β-Actin rabbit antibody (1:5,000, ABclonal, China).

Techniques: Kinase Assay, Staining, Software, Western Blot, Quantitation Assay

Journal: Frontiers in Physiology

Article Title: TRPC6 mediates high glucose-induced mitochondrial fission through activation of CDK5 in cultured human podocytes

doi: 10.3389/fphys.2022.984760

Figure Lengend Snippet: Effects of HG treatment on TRPC6 expression and TRPC6-dependent Ca 2+ influx. (A) The mRNA expression levels of TRPC6 in the control, HM, and HG groups ( n = 3 independent experiments). (B) Representative Western blot and quantitation analysis of TRPC6 in the control, HM, and HG groups ( n = 3 independent experiments). (C) The mRNA expression levels of TRPC6 in the control, scramble, and siTRPC6 groups ( n = 3 independent experiments). (D) Representative Western blot and quantitation analysis of TRPC6 in the control, scramble, and siTRPC6 groups ( n = 3 independent experiments). (E–G) Confocal microscopy using Fluo-3 fluorescence methods was used to observe changes in the intracellular Ca 2+ concentration. (E) Representative traces (left) and summary data (right) of the OAG-induced intracellular Ca 2+ changes in the HG-treated podocytes, incubated with or without 1.8 mM Ca 2+ ( n = 3 independent experiments). (F) Representative traces (left) and summary data (right) of the TG-induced SOCE (phase Ⅰ) and OAG-induced ROCE (phase Ⅱ) in the control, HM, and HG groups ( n = 3 independent experiments). (G) Representative traces (left) and summary data (right) of the TG-induced SOCE (phase Ⅰ) and OAG-induced ROCE (phase Ⅱ) in the HG + scramble and HG + siTRPC6 groups ( n = 3 independent experiments) ns, no statistical significance. * p

Article Snippet: Western blotting was performed as described previously ( ) and probed with the following primary antibodies: TRPC6 polyclonal rabbit antibodies (1:200, Alomone Labs, Israel), calpain-1 monoclonal rabbit antibodies (1:1,000, Abcam, United States), Drp1 monoclonal rabbit antibodies, p-Drp1 polyclonal rabbit antibodies, Mfn2 monoclonal rabbit antibodies, Opa1 monoclonal rabbit antibodies (1:1,000, Cell Signaling Technology, United States), or β-Actin rabbit antibody (1:5,000, ABclonal, China).

Techniques: Expressing, Western Blot, Quantitation Assay, Confocal Microscopy, Fluorescence, Concentration Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation

doi: 10.1016/j.jbc.2021.101497

Figure Lengend Snippet: Effect of FS48 on the secretion of TNF-α and IL-2 in Jurkat T cells stimulated with PMA/ionomycin. A , effect of FS48 (1, 3, 10, 30 μM) and 100 nM MgTx on the proliferation of Jurkat T cells stimulated with PMA/ionomycin. B , knockdown of Kv1.3 channel expression with different siRNA. C and D , the mRNA production of TNF-α and IL-2. E and F , the inhibition rate of TNF-α and IL-2 secretion in Jurkat T cells. G and H , the inhibition rate of TNF-α and IL-2 in Jurkat T cells after knockdown Kv1.3. All data are presented as mean ± SD (n ≥ 3). ### p

Article Snippet: Primary antibodies against Kv1.3 (4 °C, 16 h, 1: 200), p65, p38, p-p38, ERK, p-ERK, JNK, p-JNK, Histone H3, NF-кB p65, NFATc1, GAPDH, (4 °C, 16 h, 1:1000), and horseradish peroxidase–conjugated secondary antibodies (26 °C, 1 h, 1:2000) were applied, respectively.

Techniques: Expressing, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation

doi: 10.1016/j.jbc.2021.101497

Figure Lengend Snippet: Effects of FS48 on mRNA and protein expression of Kv1.3 channel. A , the viability of Jurkat T cells incubated with indicated concentrations of FS48 for 24 h. B , the relative expression analysis of KCNA3 mRNA in the presence and absence of FS48 and MgTx by qRT-PCR. C , Kv1.3 protein expression analysis of Kv1.3 channel. The cells were treated with PMA/ionomycin (50 ng/ml; 1 μg/ml) for 24 h after incubated with 3 μM FS48 and 100 nM MgTx for 1 h and then were collected for Western blot analysis. D , the ratios of Kv1.3 proteins to GAPDH. Quantity One software (Bio-Rad) was used for band density analysis. Data are shown as mean ± SD (n ≥ 3). # p

Article Snippet: Primary antibodies against Kv1.3 (4 °C, 16 h, 1: 200), p65, p38, p-p38, ERK, p-ERK, JNK, p-JNK, Histone H3, NF-кB p65, NFATc1, GAPDH, (4 °C, 16 h, 1:1000), and horseradish peroxidase–conjugated secondary antibodies (26 °C, 1 h, 1:2000) were applied, respectively.

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation

doi: 10.1016/j.jbc.2021.101497

Figure Lengend Snippet: Modulation of FS48 on endogenous voltage-gated potassium channels. A , representative traces of MgTx and different concentrations of FS48 suppressing the Kv1.3 currents in Jurkat T cells. Currents were elicited by applying 200 ms depolarization pulses from a holding potential of −70 mV to +40 mV in Jurkat T cells. B , concentration–response curve of FS48 inhibiting Kv1.3 currents in Jurkat T cells. Currents were normalized to the control and fitted by a Hill equation. C , current–voltage relationships (I-V). Test potentials were ranged from −50 mV to +40 mV with 10 mV increment steps. Y-axis represents the currents at different activation potential and normalized to the bath current at +40 mV in the present ( red ) or absent ( black ) of FS48; The solid lines represent the average Boltzmann sigmoidal fits. Data are shown as mean ± SD (n ≥ 3). ∗ p

Article Snippet: Primary antibodies against Kv1.3 (4 °C, 16 h, 1: 200), p65, p38, p-p38, ERK, p-ERK, JNK, p-JNK, Histone H3, NF-кB p65, NFATc1, GAPDH, (4 °C, 16 h, 1:1000), and horseradish peroxidase–conjugated secondary antibodies (26 °C, 1 h, 1:2000) were applied, respectively.

Techniques: Concentration Assay, Activation Assay