Journal: Frontiers in Pharmacology

Article Title: CyPPA, a Positive SK3/SK2 Modulator, Reduces Activity of Dopaminergic Neurons, Inhibits Dopamine Release, and Counteracts Hyperdopaminergic Behaviors Induced by Methylphenidate ¹

doi: 10.3389/fphar.2012.00011

Figure Lengend Snippet: CyPPA inhibits spontaneous and evoked release of dopamine form TH/SK3 positive cultured rat midbrain neurons . (A) Western blot detection of SK3 in rat midbrain cultures. Extracts of HEK293 cells transiently transfected with rSK1, rSK2, and rSK3 and adult rat brain membranes (RBM) as well as lysates from cultured midbrain neurons were separated on 4–15% SDS-PAGE and immunoblotted with anti-SK3 antibody. A band of approximately 70 kDa was detected in midbrain cultures, RBM lysates, and HEK293 cells transfected with rSK3. This band was absent in lanes loaded with HEK293 cells expressing rSK1 and rSK2. (B) Immunodetection of SK3 subunits in rat midbrain cultures. Cultured midbrain neurons (8 days in vitro ) were double-labeled for SK3 and tyrosine hydroxylase (TH). In TH-positive dopaminergic neurons, the SK3 subunit displayed a primarily somatic–dendritic localization whereas little immunoreactivity was associated with axons (arrow). Scale bar 20 μm. (C) Basal release and stimulated release with 25 mM KCl or inhibited release with 30 μM Cd 2+ and with 1 μM TTX. (D) Basal release and stimulated release with 3 μM haloperidol and inhibited release with 10 μM quinpirole. Blue bars represent inhibited release with increasing concentrations of CyPPA (* P < 0.05, ** P < 0.01). The data presented are representative of three independent experiments.

Article Snippet: Neurobasal media and B-27 were obtained from Invitrogen (Nærum, Denmark) and 3 H-DA was acquired from PerkinElmer Life and Analytical Sciences (Boston, MA, USA) The primary antibodies used were a rabbit polyclonal anti-SK3 antibody (1:100, APC-025) from Alomone Labs (Jerusalem, Israel) and a monoclonal mouse anti-tyrosine hydroxylase antibody (1:150, clone LNC1) from Millipore (Copenhagen, Denmark).

Techniques: Cell Culture, Western Blot, Transfection, SDS Page, Expressing, Immunodetection, In Vitro, Labeling, Immunofluorescence

Journal:

Article Title:

doi:

Figure Lengend Snippet: Specific primers for three SKCa isoforms (SK1-3) amplification from mRNA of isolated guinea pig ventricular myocyte.

Article Snippet: One membrane was incubated with specific primary antibody anti-SK3 (Alomone Lab APC-103) (SK3 left panel); another membrane was incubated with the same antibody, anti-SK3, but which was neutralized with the corresponding immunizing SK3 peptide before being added (SK3 preincubation, right panel).

Techniques: Amplification, Isolation

Journal:

Article Title:

doi:

Figure Lengend Snippet: Expression of mSKCa channels in guinea pig hearts. A: A representative agarose gel analysis of RT-PCR amplified SK1, SK2 and SK3 from guinea pig cardiomyocytes (lanes 1–6) and human ventricular tissue (lanes 9–10). Lane 1: SK1-N; Lane 2: SK1-C; Lane 3: SK2-N; Lane 4: SK2-C; Lane 5: SK3-N; Lane 6: SK3-C; Lane 9: human SK3-N; Lane 10: human SK3-C; lanes 7 and 8: DNA ladder; Lane 11: RNA control. B: Alignment of SK3 splice variants. Red: deduced amino acid (a.a.) sequences from amplified genes; Blue: P-W sequence designating SK3.2; Black dashes: guinea SK3 transmembrane (T) domains 1–6; CaMBD: Ca2+ calmodulin binding domain; Bold: human vs. guinea pig a.a. differences; Yellow: truncated N terminus (a.a. 1–277) or C-terminus (a.a. 627–720) of SK3.1 as overexpressed in HL-1 cells (Fig. 8). C: Western blot analyses of SK3 in guinea pig heart (Heart), heart mitochondria (Mito) and mitochondrial inner membrane (IMM). One membrane was incubated with specific primary antibody anti-SK3 (Alomone Lab APC-103) (SK3 left panel); another membrane was incubated with the same antibody, anti-SK3, but which was neutralized with the corresponding immunizing SK3 peptide before being added (SK3 preincubation, right panel). Tubulin, GM130, and calnexin were used to monitor purity of mitochondria and IMM; no contamination by cytosol, Golgi, or sarcoplasmic reticular membrane protein was observed in mitochondria and IMM. ANT was used to monitor integrity of mitochondria and IMM. Since SK3 expression is weak in mitochondria, the protein concentration loaded in the mito lane was larger than that in heart and IMM lanes.

Article Snippet: One membrane was incubated with specific primary antibody anti-SK3 (Alomone Lab APC-103) (SK3 left panel); another membrane was incubated with the same antibody, anti-SK3, but which was neutralized with the corresponding immunizing SK3 peptide before being added (SK3 preincubation, right panel).

Techniques: Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Binding Assay, Western Blot, Incubation, Protein Concentration

Journal:

Article Title:

doi:

Figure Lengend Snippet: Representative recordings of mK+ uptake activated by adding CaCl2 to buffer in HL-1 cells transfected with SK3.1 constructs. Traces of PBFI fluorescence indicate changes in m [K+]. A: in SK3FL-EGFP transfected HL-1 cells; B: in SK3FL-EGFP transfected HL-1 cells with alamethicin, a mitochondrial pore forming peptide; C: in pEGFP-N3 transfected HL-1 cells; D: in SK3Δ626–720–EGFP transfected HL-1 cells; and E: in SK3FL-EGFP transfected HL-1 cells with apamin, a blocker of the SK3 channel. F: averaged ΔPBFI ratios induced by adding CaCl2 to permeabilized HL-1 cells. n= 20 cells; *P < 0.05 vs. EGFP control. Because of the presence of 3 mM EGTA and 5 mM MgCl2, mitochondrial [Ca2+] with added 2 mM CaCl2 remained within the nM range.

Article Snippet: One membrane was incubated with specific primary antibody anti-SK3 (Alomone Lab APC-103) (SK3 left panel); another membrane was incubated with the same antibody, anti-SK3, but which was neutralized with the corresponding immunizing SK3 peptide before being added (SK3 preincubation, right panel).

Techniques: Transfection, Construct, Fluorescence

Journal:

Article Title:

doi:

Figure Lengend Snippet: Location of mSK3 in human heart and rat cardiomyocytes. A: Immuno-electron microscopic (IEM) detection of isolated human ventricular mitochondria immunostained with specific antibody against SK3. B: Western blot analyses of SK3 expression in human ventricular tissue (heart), mitochondria (mito) and inner mitochondrial membrane (IMM). Tubulin confirmed purity of mitochondrial isolation, adenine nucleotide translocase (ANT) confirmed enrichment of mitochondria and IMM, and GM130 and calnexin confirmed absence of contamination by Golgi or sarcoplasmic reticulum membrane, respectively. C: Confocal fluorescence images of adult rat cardiomyocytes immuno-stained simultaneously with SK3 antibody followed by FITC conjugated secondary antibody (middle panel, Green) and COX 1 (cytochrome c oxidase) antibody, followed by Alexa Fluor 546 conjugated secondary antibody (top panel, red). Yellow color in merged image (bottom panel) depicts SK3 localized in mitochondria; the intensity profile panel showed the overlapping degree of SK3 with COX 1. The x-axis in the intensity profile is from top to bottom. The yellow rectangles label the overlap areas of SK3 with COX 1.

Article Snippet: One membrane was incubated with specific primary antibody anti-SK3 (Alomone Lab APC-103) (SK3 left panel); another membrane was incubated with the same antibody, anti-SK3, but which was neutralized with the corresponding immunizing SK3 peptide before being added (SK3 preincubation, right panel).

Techniques: Isolation, Western Blot, Expressing, Fluorescence, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: SK3.1 localized into mitochondria. Top panels: Schematic of SK3FL-EGFP, SK3Δ1–277–EGFP and SK3Δ626–720–EGFP constructs. Q sequence, poly glycine sequence; T1–6, transmembrane regions; P, selectivity pore; CaMBD, Ca2+ calmodulin binding domain; EGFP, enhanced green fluorescence protein. Bottom panels: Confocal fluorescence images of HL-1 cells transfected with SK3FL-EGFP (A, Green), SK3Δ626–720–EGFP (B, Green) or SK3Δ1–277–EGFP (C, Green) and stained with Mitotracker red, (MTR, Red). Merging (orange) depicts colocalization of SK3 with MTR; the intensity profiles show the overlapping degree of SK3 with MTR. The x-axis in the intensity profile was recorded from top to bottom and from left to right for regions 1 and 2 respectively. The yellow rectangles label the overlap areas of SK3 with MTR.

Article Snippet: One membrane was incubated with specific primary antibody anti-SK3 (Alomone Lab APC-103) (SK3 left panel); another membrane was incubated with the same antibody, anti-SK3, but which was neutralized with the corresponding immunizing SK3 peptide before being added (SK3 preincubation, right panel).

Techniques: Construct, Sequencing, Binding Assay, Fluorescence, Transfection, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: Lactate dehydrogenase (LDH) release is reduced if SK3 C-terminus is intact. HL-1 cells transfected with pEGFP-N3, SK3FL, SK3Δ1–277 and SK3Δ626–720 were subjected to hypoxia/reoxygenation or normoxia. LDH activity was measured in the reoxygenation medium. Data presented are means ± SE from 3 independent experiments. *P < 0.05 vs. normoxia groups, #P < 0.05 vs. EGFP groups.

Article Snippet: One membrane was incubated with specific primary antibody anti-SK3 (Alomone Lab APC-103) (SK3 left panel); another membrane was incubated with the same antibody, anti-SK3, but which was neutralized with the corresponding immunizing SK3 peptide before being added (SK3 preincubation, right panel).

Techniques: Transfection, Activity Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: Silencing SK3 in HL-1 cells increases cell apoptosis. A: Real-time PCR assessment of SK3 relative mRNA abundance in HL-1 cells transfected with SK3 silence siRNA or scramble siRNA. Data presented are means ± SE from 3 independent experiments. * P < 0.05 vs. scramble siRNA groups. B: Representative TUNEL staining of HL-1 cells transfected with SK3 siRNA or scramble siRNA and subjected to normoxia (control) or to 2 h hypoxia and 16 h reoxygenation. Apoptotic nuclei were TUNEL stained (red) and counterstained with DAPI (blue) to label nuclei. C: Quantitative analysis of TUNEL-positive HL-1 cells. Mean ± SE, n = 2000 HL-1 cells per group. * P < 0.05 vs. scramble siRNA, # P < 0.05 vs. normoxia.

Article Snippet: One membrane was incubated with specific primary antibody anti-SK3 (Alomone Lab APC-103) (SK3 left panel); another membrane was incubated with the same antibody, anti-SK3, but which was neutralized with the corresponding immunizing SK3 peptide before being added (SK3 preincubation, right panel).

Techniques: Real-time Polymerase Chain Reaction, Transfection, TUNEL Assay, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: Silencing SK3 in HL-1 cells attenuates ΔΨm. HL-1 cells were transfected with SK3 silenced siRNA or scramble siRNA. Changes in TMRM fluorescence (ΔΨm) was measured in HL- 1 cells subjected to simulated ischemia (blocked respiration). A: Time course of relative changes in ΔΨm was assessed in SK3 siRNA (n = 40 cells) or in scramble siRNA (n =50 cells) transfected HL-1 cells exposed to 10 min simulated ischemia followed by 8 min resuffusion of cells with normal Tyrode to wash out the simulated ischemia buffer and restore control conditions. (Time control = no ischemia; n =45 cells). *P < 0.05 vs. scramble siRNA. B: Representative confocal images of TMRM fluorescence in HL-1 cells transfected with SK3 siRNA or scramble siRNA at conditions and times noted in A.

Article Snippet: One membrane was incubated with specific primary antibody anti-SK3 (Alomone Lab APC-103) (SK3 left panel); another membrane was incubated with the same antibody, anti-SK3, but which was neutralized with the corresponding immunizing SK3 peptide before being added (SK3 preincubation, right panel).

Techniques: Transfection, Fluorescence

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Concentration Assay, Bioprocessing

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Characterization of the molecular interactions between Cav1.3, SK3, and HCN channels using co-immunoprecipitation and immunoblot. Proteins were membrane-extracted in CL47a solubilization buffer (IP1) or in CL48 buffer (IP2) and immunoprecipitated using anti-Cav1.3 ( A ), anti-SK3 ( B ), and anti-Kv4.3 antibodies ( C ). Blots were performed on starting material, P2 membrane fraction (P2), or on solubilized P2 proteins (sol) or after immunoprecipitation (IP1, IP2). Blots were probed with anti-SK3 (( A ), right panel), anti-Cav1.3 (( B ), right panel), anti-HCN4 (( A , B ) left panels; ( C ), right panel), anti-Kv4.3 (( C ), left panel), and anti-HCN2 (( C ), middle panel). P2: enriched P2 membrane fraction. IP1: immunoprecipitated proteins in CL47a buffer. IP2: immunoprecipitated proteins in CL48 buffer. sol: solubilisate. WB: Western blot (immunoblot).

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Membrane, Immunoprecipitation

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Schematic representation of midbrain ion channel interactions detected through co-immunoprecipitation and immunoblot. Lines indicate co-immunoprecipitated ion channels. The arrows point from the bait to the prey revealed through immunoblot.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Protein nano-environment of midbrain ion channels determined using affinity-purification mass spectrometry. ( A ) Left panel, Cav1.2 subunit ( Cacna1c gene) immunoprecipitations. Right panel, Cav1.3 subunit ( Cacna1d gene) immunoprecipitation. ( B ) SK3 subunit ( Kcnn3 gene) immunoprecipitation. ( C ) Left panel, HCN2 subunit immunoprecipitation. Right panel, HCN4 subunit immunoprecipitation. ( D ) Kv4.3 ( Kcnd3 gene) immunoprecipitation. High-confidence Class A (solid line) and low-confidence Class B (dashed lines) thresholds are displayed on the plots. Plain squares represent the bait and empty square interactants. The most relevant of the significant interactants are named.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Affinity Purification, Mass Spectrometry, Immunoprecipitation

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Visualization of the mass-spectrometry-based protein interaction network. The 6 ion channels of interest are depicted using different colors (blue, Kv4.3; pink and red, HCN2 and HCN4; yellow and orange, Cav1.2 and Cav1.3; green, SK3). Interactions are shown with lines of according colors. Thick lines represent Class A interactions, thinner lines Class B. Putative interactants were compared with the CRAPome 2.0 database (Contaminant Repository for Affinity Purification). Proteins identified in less than 1% of all CRAPome negative experiments were considered highly specific (black circles), between 1 and 2%, specific (grey circles), and between 2 and 10%, weakly specific (white circles) interactants.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Mass Spectrometry, Affinity Purification

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Cav1.3-HCN2-SK3 complex characterization in SNc DA neurons using proximity ligation assay. ( A ) Left, representative images of the PLA reaction (green) in wild-type (Wt) or Cav1.3 −/− SNc DA neurons using anti-Cav1.3 and anti-HCN2 antibodies. Middle, immunolabeling of SNc DA neurons with anti-TH antibodies (red). Right, merged images. Scale bars, 10 μm. ( B ) Representative images of the PLA reaction (green) in wild-type (Wt) SNc DA neurons using anti-SK3 and anti-HCN2 antibodies (top row) or anti-SK2 and anti-HCN2 antibodies (bottom row). Middle, immunolabeling of SNc DA neurons with anti-TH antibodies (red). Right, merged images. Scale bars, 10 μm. ( C ) Quantification of PLA fluorescent puncta on SNc DA neurons. Left, Cav1.3 and HCN2 PLA from 3 Wt (empty black circles; n = 21 images) or 3 Cav1.3 −/− (empty red circles; n = 18 images) mice. Superimposed box and whiskers plots allow us to visualize the median and interquartile range. Middle, HCN2 and SK3 PLA (empty black circles; n = 11 images) or HCN2 and SK2 (empty orange circles; n = 7 images) from 2 independent experiments or anti-Kv4.3 and HCN2 (empty grey circle; n = 4 images) PLA from 1 experiment. Superimposed box and whiskers plots allow us to visualize the median and interquartile range. Right, Cav1.3 and SK3 PLA from 2 Wt (empty black circles; n = 10 images) or 2 Cav1.3 −/− (empty red circles; n = 8 images) mice. Superimposed box and whiskers plots allow us to visualize the median and interquartile range. ** p < 0.005; *** p < 0.001, Wilcoxon–Mann–Whitney test.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Proximity Ligation Assay, Immunolabeling, MANN-WHITNEY, Immunofluorescence

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Characterization of the interactions between SK3 and Tsc1 in SNc DA neurons using proximity ligation assay. ( A ) Left, representative images of the PLA reaction (green) in SNc DA neurons using anti-Tsc1 and anti-SK3 antibodies (top row) or anti-Tsc1 and anti-SK2 antibodies (bottom row). Middle, immunolabeling of SNc DA neurons with anti-TH antibodies (red). Right, merged images. Scale bars, 10 μm. ( B ) Quantification of PLA fluorescent puncta in SNc DA neurons using anti-Tsc1 and SK3 antibodies (empty black circles; n = 16 images) or anti-Tsc1 and SK2 antibodies (empty orange circles; n = 14 images) from 2 independent experiments. Superimposed box and whiskers plots allow us to visualize the median and interquartile range. ** p < 0.005, Wilcoxon–Mann–Whitney test.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Proximity Ligation Assay, Immunolabeling, MANN-WHITNEY, Immunofluorescence

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Schematic representation summarizing the ion channel interactions detected with co-immunoprecipitation, mass spectrometry and proximity ligation assay. Plain lines indicate co-immunoprecipitated ion channels. Dashed lines indicate interactions detected in mass spectrometry experiments. The arrows point from the bait to the prey. Dotted lines link proteins detected in close proximity using PLA in situ in SNc DA neurons.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Proximity Ligation Assay, Immunoprecipitation, In Situ, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Western Blot

Journal: bioRxiv

Article Title: Safety and efficacy of C9ORF72 -repeat RNA nuclear export inhibition in amyotrophic lateral sclerosis

doi: 10.1101/2021.04.12.438950

Figure Lengend Snippet: Relative expression levels of KCNN1 , KCNN2 , KCNN3 and KCNN4 mRNAs were quantified using qRT-PCR in biological triplicates following normalization to U1 snRNA levels and to 100% for healthy neurons treated with Ctrl-RNAi (mean ± SEM; two-way ANOVA with Tukey’s correction for multiple comparisons; NS: not significant; ***: p<0.001, ****: p<0.0001; N (qRT-PCR reactions)=3).

Article Snippet: Primers for Drosophila Tub84b (Ling et al ., PLoS ONE 2011; 6:e17762): Fwd: 5’-TGGGCCCGTCTGGACCACAA-3’ Rev: 5’-TCGCCGTCACCGGAGTCCAT-3’ Primers for Drosophila SK (designed using Primer-BLAST): Fwd: 5’-ACCCTGTACTGCTGTTGCC-3’ Rev: 5’-TGTACAGATTCTGATGGATGGCTT-3’ Primers for Drosophila NAAT1 (designed using Primer-BLAST): Fwd: 5’-CACGGGATTGGCCTTCATCT-3’ Rev: 5’-CACGGGATTGGCCTTCATCT-3’ Primers for Drosophila DHD (designed using Primer-BLAST): Fwd: 5’-GTGGTCCCTGCAAGGAAATG-3’ Rev: 5’-CACCTTGTAGCGCTCCGTC-3’ Primers for Human U1 snRNA (Hautbergue et al ., 2017; 8:16063): Fwd: 5’-CCATGATCACGAAGGTGGTT-3’ Rev: 5’-ATGCAGTCGAGTTTCCCACA-3’ Primers for Human SRSF1 (Hautbergue et al ., 2017; 8:16063): Fwd: 5’-CCGCATCTACGTGGGTAACT-3’ Rev: 5’-TCGAACTCAACGAAGGCGAA-3 Primers for Human C9ORF72 (Hautbergue et al ., 2017; 8:16063): Intron-1 Rev: 5’-GGAGAGAGGGTGGGAAAAAC-3’ Exon-3 Rev: 5’-GTCGACATGACTGCATTCCA-3’ Exon-1 For: 5’-TCAAACAGCGACAAGTTCCG-3’ Primers for Human Usp49 (Origene): Fwd: 5’-GGAGAATCTACGCTTGTGACCAG-3’ Rev: 5’-CGGAGAACCTGAGGTAGTCTGT-3’ Primers for Human RSL1D1 (Origene): Fwd: 5’-TCCGAAGACGAAATCCCACAGC-3’ Rev: 5’-GTGCTGGGATTAGGACTCTTTGC-3’ Primers for Human MSH6 (Origene): Fwd: 5’-AAGGACTGGCAGTCTGCTGTAG-3’ Rev: 5’-CGGCAACACAGAATTACTGGGCGA-3’ Primers for Human RBM15 (Origene): Fwd: 5’-CTTCCCACCTTGTGAGTTCTCC-3’ Rev: 5’-CTTCTTGTTCTCATACCTAACTCC-3’ Primers for Human MTCL3 (Origene): Fwd: 5’-TGCTCAAGTGCCGTCTGGAACA-3’ Rev: 5’-TGACTGTCTGCCAGGAGCTTCT-3’ Primers for Human NUP98 (Origene): Fwd: 5’-CCATCTATGGATGACCTGTAAA-3’ Rev: 5’-TCCGACCAATAGTGAAATCAGAGA-3’ Primers for Human DAPK1 (Origene): Fwd: 5’-CCAGACTGTCTTCCACCAACTC-3’ Rev: 5’-TCCTCACACTCACGTTCTCGCA-3’ Primers for Human FN1 (Origene): Fwd: 5’-GGACACAACGATGCTTCCTGAG-3’ Rev: 5’-ACAACACCGAGGTGACTGAGAC-3’ Primers for Human USP19 (Origene): Fwd: 5’-GCTGCTATCCTCAGAGTTGGCT-3’ Rev: 5’-TCATCCTCCGACTGTTGCTTCC-3’ Primers for Human KCNN1 (Origene): Fwd: 5’-TGCTGGTCTTCAGCATCTCCTC-3’ Rev: 5’-CGTAGCCAATGGAGAGGAAGGT-3’ Primers for Human KCNN2 (Origene): Fwd: 5’-GCCTTATCAGTCTCTCCACGATC-3’ Rev: 5’-CCAGTCATCTGCTCCATTGTCC-3’ Primers for Human KCNN3 (Origene): Fwd: 5’-GCCTTATCAGTCTGTCCACCATC-3’ Rev: 5’-TACAGGATGCGCTCGTAGGTCA-3’ Primers for Human KCNN4 (Origene): Fwd: 5’-CATTCCTGACCATCGGCTATGG-3’ Rev: 5’-GCCTTGTTAAACTCCAGCTTCCG-3’ Primers for Human TRX1 (Origene): Fwd: 5’-GTAGTTGACTTCTCAGCCACGTG-3’ Rev: 5’-CTGACAGTCATCCACATCTACTTC-3’

Techniques: Expressing, Quantitative RT-PCR

Journal: Bioactive Materials

Article Title: A continuous adhesion-enhanced osteogenic pathway in artificial scaffold drives cellular infiltration and condensed mineralization for rapid bone regeneration

doi: 10.1016/j.bioactmat.2026.02.026

Figure Lengend Snippet: IHC and IF staining of functional proteins. (a) H&E and Masson's staining of defect area after implantation of CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for one month. (b) OCN IHC and IF staining of CPH/rGO-3/0 and CPH/rGO-3/0.6 after implantation for one month and the corresponding OD value and fluorescence intensity statistics. (c) IF staining of FREM1 and ANK3 in scaffolds after implanting for one months in rats and corresponding fluorescence intensity statistics. (d) IF staining of KCNN3 and KCNH1 in scaffolds after implanting for one months in rats and corresponding fluorescence intensity statistics. n = 20 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001.

Article Snippet: Hematoxylin-Eosin (Solarbio, Beijing, China) Stain Kit, Masson's Trichrome Stain Kit (Solarbio, Beijing, China), OCN monoclonal antibody (ThermoFisher Scientific, USA) FREM1 and KCNN3 antibody (Bioss Antibodies, Beijing, China), ANK3 antibody (Proteintech, Wuhan, China), KCNH1 (Servicecbio, Wuhan, China) were used to stain the slice of specimens.

Techniques: Staining, Functional Assay, Fluorescence

Journal: Biochemical and biophysical research communications

Article Title: Role of SK channel activation in determining the action potential configuration in freshly isolated human atrial myocytes from the SKArF study.

doi: 10.1016/j.bbrc.2019.03.074

Figure Lengend Snippet: Fig. 3. Presence of SK2 and SK3 channel subunits in human atrial myocytes. A. Confocal images (0.5-mm section) of a fixed and permeabilized cell labelled with anti-SK2 (green) and anti-SK3 (red). Labelling was observed at the cell periphery and superimposition of both images showed a significant degree of colocalization (yellow). B. Graph illustrates that an apparent similar level of labelling of SK2 and SK3 subunits in acutely isolated human atrial myocytes. C. Confocal images of SK2 (green) and SK3 (red) showing labelling at the cell edge. Superimposition of SK2 and SK3 channel subunit labelling demonstrates significant colocalization (yellow), which was quantified by using Mander's coefficient. Measured SK2 and SK3 labelling gave a Mander's coefficient of co-localization of SK3 overlapping SK2 by 0.94 ± 0.05 and SK2 overlapping SK3 by 093 ± 0.03. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cells were then incubated overnight at room temperature with mouse anti-SK2 antibody (Biorbyt: orb333955) and rabbit anti-SK3 antibody (Biorbyt: orb157735), added as a 1:200 dilution in blocking solution of composition (mM): phosphate buffer saline, bovine serum albumin (Sigma Aldrich, A9418) (1%), and foetal calf serum (Thermo Fisher Scientific, 10500-064) (2%) [20,21].

Techniques: Isolation