Journal: Biochimica et biophysica acta

Article Title: Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

doi: 10.1016/j.bbabio.2017.03.005

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: Biochimica et biophysica acta

Article Title: Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

doi: 10.1016/j.bbabio.2017.03.005

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: Biochimica et biophysica acta

Article Title: Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

doi: 10.1016/j.bbabio.2017.03.005

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: Biochimica et biophysica acta

Article Title: Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

doi: 10.1016/j.bbabio.2017.03.005

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: Biochimica et biophysica acta

Article Title: Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

doi: 10.1016/j.bbabio.2017.03.005

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: Biochimica et biophysica acta

Article Title: Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

doi: 10.1016/j.bbabio.2017.03.005

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: Biochimica et biophysica acta

Article Title: Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

doi: 10.1016/j.bbabio.2017.03.005

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: Biochimica et biophysica acta

Article Title: Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

doi: 10.1016/j.bbabio.2017.03.005

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: eLife

Article Title: Single-cell glycomics analysis by CyTOF-Lec reveals glycan features defining cells differentially susceptible to HIV

doi: 10.7554/eLife.78870

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Human CD4 (SK3) (Mouse, Monoclonal) , Fluidigm , Cat# 3174004B , CyTOF (1:50).

Techniques: Recombinant, Plasmid Preparation, Software

Journal: Cancers

Article Title: Tonsillar Cancer with High CD8 + T-Cell Infiltration Features Increased Levels of Dendritic Cells and Transcriptional Regulation Associated with an Inflamed Tumor Microenvironment

doi: 10.3390/cancers13215341

Figure Lengend Snippet: Antibody panel employed for leukocyte quantification.

Article Snippet: CD4–BV510 , SK3 , BD Biosciences.

Techniques: