Structured Review

Millipore antibodies against kv1 3
Analysis of <t>Kv1.3</t> activation kinetics. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (-) free KCNE4. Different combinations yielded fixed or putative Kv1.3:KCNE4 stoichiometries. Cells were held at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. Peak currents from −20 mV to +60 mV were calculated. ( A ) Time to reach the peak current in the open channels from −20 mV to +60 mV. The time scale is presented in log values for easier viewing. ( B ) Time to peak at 0 mV. Values are the means ± SE of 8–14 cells; * p
Antibodies Against Kv1 3, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/antibodies against kv1 3/product/Millipore
Average 93 stars, based on 2 article reviews
Price from $9.99 to $1999.99
antibodies against kv1 3 - by Bioz Stars, 2022-09
93/100 stars

Images

1) Product Images from "Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex"

Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

Journal: Cells

doi: 10.3390/cells9051128

Analysis of Kv1.3 activation kinetics. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (-) free KCNE4. Different combinations yielded fixed or putative Kv1.3:KCNE4 stoichiometries. Cells were held at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. Peak currents from −20 mV to +60 mV were calculated. ( A ) Time to reach the peak current in the open channels from −20 mV to +60 mV. The time scale is presented in log values for easier viewing. ( B ) Time to peak at 0 mV. Values are the means ± SE of 8–14 cells; * p
Figure Legend Snippet: Analysis of Kv1.3 activation kinetics. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (-) free KCNE4. Different combinations yielded fixed or putative Kv1.3:KCNE4 stoichiometries. Cells were held at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. Peak currents from −20 mV to +60 mV were calculated. ( A ) Time to reach the peak current in the open channels from −20 mV to +60 mV. The time scale is presented in log values for easier viewing. ( B ) Time to peak at 0 mV. Values are the means ± SE of 8–14 cells; * p

Techniques Used: Activation Assay, Transfection

KCNE4 specifically modulates Kv1.3. HEK-293 cells were transfected with Kv1.3-YFP in the presence (+KCNE4) or the absence of KCNE4-CFP. Cells were held at −80 mV, and currents were elicited by 1 s depolarizing pulses from −60 mV to +60 mV in 10 mV intervals. ( A ) Representative traces at +60 mV. ( B ) Current density vs. the voltage of K + currents ( p
Figure Legend Snippet: KCNE4 specifically modulates Kv1.3. HEK-293 cells were transfected with Kv1.3-YFP in the presence (+KCNE4) or the absence of KCNE4-CFP. Cells were held at −80 mV, and currents were elicited by 1 s depolarizing pulses from −60 mV to +60 mV in 10 mV intervals. ( A ) Representative traces at +60 mV. ( B ) Current density vs. the voltage of K + currents ( p

Techniques Used: Transfection

GFP bleaching steps from HEK-293 cells transfected with Kv1.3 in the absence or presence of KCNE4. ( A – C ) Cells were transfected with Kv1.3-loopBAD-GFP. ( A ) Representative 1.5 s and 6.5 s time-lapse snapshots from the TIRFM video (488 nm laser). White arrow heads (a, b) point to the yellow square ROI (6 × 6 pixels) in panel c. (a) Initial fluorescence intensity; (b) fluorescence intensity after the first bleaching step at the same spot; (c) merge image. Yellow square indicates the ROI. The white line delineates the cell shape. Scale bar, 5 μm. ( B ) Representative graph of the bleaching steps for the different spots analyzed. Red arrows point to 4 bleaching steps. Squares, at the right, represent the fluorescence intensity after 4 consecutive bleaching steps at the same ROI. 0 s, initial fluorescence; 43.9 s, background intensity remaining after 4 consecutive bleaching steps. ( C ) Relative frequency of 1–4 bleaching events counted per spot. The black bars correspond to the experimental frequencies observed. Red dashed lines correspond to the theoretical distributions of bleaching steps with p = 0.67. ( D – G ) GFP bleaching steps from HEK-293 cells transfected with KCNE4-loopBAD-GFP and Kv1.3-Apple. ( D ) Representative snapshots from a cropped TIRF microscopy video. Upper panel (561 nm laser): Kv1.3-Apple. Lower panel (488 nm laser): KCNE4-loop-BADGFP. Merge panel zooms squares from previous panels. White squares ROIs (6 × 6 pixels) indicate colocalizing stationary spots. Putative oligomeric composition of the Kv1.3/KCNE4 complex is represented with circles at the left. While 4 white circles indicate the Kv1.3 tetramer, KCNE4 units are represented with gray and black circles. ( E – G ) Representative graph of the GFP bleaching steps from the different spots analyzed. ( E ) Two bleaching steps indicating a 4:2 Kv1.3:KCNE4 stoichiometry. ( F ) Three bleaching steps suggesting a 4:3 Kv1.3:KCNE4 ratio. ( G ) Four bleaching steps highlighting a 4:4 Kv1.3:KCNE4 stoichiometry. ( H ) Histogram with the relative frequency of 1–4 bleaching events counted for spot. Bars correspond to the experimental frequencies observed. ( I ) Histograms representing the theoretical distribution of the bleaching events when KCNE4 is present at the Kv1.3/KCNE4 complex as a monomer (4:1 stoichiometry), dimer (4:2), trimer (4:3) or tetramer (4:4) with p = 0.67. In contrast to the functional Kv1.3 tetramers ( C ), no expected KCNE4 distribution fit with the distribution observed during the analysis of the experimental data.
Figure Legend Snippet: GFP bleaching steps from HEK-293 cells transfected with Kv1.3 in the absence or presence of KCNE4. ( A – C ) Cells were transfected with Kv1.3-loopBAD-GFP. ( A ) Representative 1.5 s and 6.5 s time-lapse snapshots from the TIRFM video (488 nm laser). White arrow heads (a, b) point to the yellow square ROI (6 × 6 pixels) in panel c. (a) Initial fluorescence intensity; (b) fluorescence intensity after the first bleaching step at the same spot; (c) merge image. Yellow square indicates the ROI. The white line delineates the cell shape. Scale bar, 5 μm. ( B ) Representative graph of the bleaching steps for the different spots analyzed. Red arrows point to 4 bleaching steps. Squares, at the right, represent the fluorescence intensity after 4 consecutive bleaching steps at the same ROI. 0 s, initial fluorescence; 43.9 s, background intensity remaining after 4 consecutive bleaching steps. ( C ) Relative frequency of 1–4 bleaching events counted per spot. The black bars correspond to the experimental frequencies observed. Red dashed lines correspond to the theoretical distributions of bleaching steps with p = 0.67. ( D – G ) GFP bleaching steps from HEK-293 cells transfected with KCNE4-loopBAD-GFP and Kv1.3-Apple. ( D ) Representative snapshots from a cropped TIRF microscopy video. Upper panel (561 nm laser): Kv1.3-Apple. Lower panel (488 nm laser): KCNE4-loop-BADGFP. Merge panel zooms squares from previous panels. White squares ROIs (6 × 6 pixels) indicate colocalizing stationary spots. Putative oligomeric composition of the Kv1.3/KCNE4 complex is represented with circles at the left. While 4 white circles indicate the Kv1.3 tetramer, KCNE4 units are represented with gray and black circles. ( E – G ) Representative graph of the GFP bleaching steps from the different spots analyzed. ( E ) Two bleaching steps indicating a 4:2 Kv1.3:KCNE4 stoichiometry. ( F ) Three bleaching steps suggesting a 4:3 Kv1.3:KCNE4 ratio. ( G ) Four bleaching steps highlighting a 4:4 Kv1.3:KCNE4 stoichiometry. ( H ) Histogram with the relative frequency of 1–4 bleaching events counted for spot. Bars correspond to the experimental frequencies observed. ( I ) Histograms representing the theoretical distribution of the bleaching events when KCNE4 is present at the Kv1.3/KCNE4 complex as a monomer (4:1 stoichiometry), dimer (4:2), trimer (4:3) or tetramer (4:4) with p = 0.67. In contrast to the functional Kv1.3 tetramers ( C ), no expected KCNE4 distribution fit with the distribution observed during the analysis of the experimental data.

Techniques Used: Transfection, Fluorescence, Microscopy, Functional Assay

Analysis of C-type inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (−) free KCNE4. Different combinations yielded fixed and putative Kv1.3:KCNE4 stoichiometries. ( A ) Representative traces from C-type inactivation recordings. An initial 100 ms pulse at −80 mV was applied prior to a 5 s depolarizing pulse at + 60 mV. ( B ) Relative intensity of voltage-dependent K + currents from all combinations during the first 2 s. ( C ) The constant (τ) of inactivation (in ms) of all groups. Values are the means ± SE, n = 8–14; * p
Figure Legend Snippet: Analysis of C-type inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (−) free KCNE4. Different combinations yielded fixed and putative Kv1.3:KCNE4 stoichiometries. ( A ) Representative traces from C-type inactivation recordings. An initial 100 ms pulse at −80 mV was applied prior to a 5 s depolarizing pulse at + 60 mV. ( B ) Relative intensity of voltage-dependent K + currents from all combinations during the first 2 s. ( C ) The constant (τ) of inactivation (in ms) of all groups. Values are the means ± SE, n = 8–14; * p

Techniques Used: Transfection

Analysis of the cumulative inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and K + currents were analyzed. ( A ) Chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), KCNE4:Kv1.3 (4:4). ( B ) Functional complexes with putative Kv1.3-KCNE4 stoichiometries due to further addition of extra free KCNE4 units. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). Cells were held at −80 mV, and a train of 25 depolarizing pulses to +60 mV for 200 ms was applied. ( C ) I/Imax vs. the pulse number. The ratio of the peak current amplitude during each pulse relative to that during the 1st pulse (I/Imax) was plotted against every pulse. ( D ) I/Imax reached at the last pulse train of depolarization. * p
Figure Legend Snippet: Analysis of the cumulative inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and K + currents were analyzed. ( A ) Chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), KCNE4:Kv1.3 (4:4). ( B ) Functional complexes with putative Kv1.3-KCNE4 stoichiometries due to further addition of extra free KCNE4 units. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). Cells were held at −80 mV, and a train of 25 depolarizing pulses to +60 mV for 200 ms was applied. ( C ) I/Imax vs. the pulse number. The ratio of the peak current amplitude during each pulse relative to that during the 1st pulse (I/Imax) was plotted against every pulse. ( D ) I/Imax reached at the last pulse train of depolarization. * p

Techniques Used: Transfection, Construct, Functional Assay

Chimeric constructs, protein expression and putative oligomeric formations. ( A ) Representative cartoon of the fusion proteins. All chimeras were tagged with either YFP or CFP. White and black barrels represent Kv1.3 peptides. Dark and light gray correspond to KCNE4 structures. In KCNE4-Kv1.3 and KCNE4-Kv1.3T, the 18 aa link is also indicated. ( B ) Western blot of the protein lysates of the nontransfected HEK-293 cells and HEK-293 cells transfected with KCNE4 and Kv1.3. ( C ) Protein levels of cells expressing Kv1.3T, KCNE4-Kv1.3T and KCNE4-Kv1.3. ( D ) Putative oligomerization of Kv1.3 and KCNE4 complexes according to the construct combination. Basic channels formed by chimeras exhibited fixed stoichiometries. The addition of free KCNE4 units yielded forced channels with putative stoichiometries. 1–4, the number of KCNE units by complex, which varied from 1 to 4. 2–4, the number of KCNE units by complex, which varied from 2 to 4. White and black circles represent Kv1.3 peptides. Light gray corresponds to KCNE4 chimeras linked to Kv1.3. Dark gray highlights excess KCNE4 units.
Figure Legend Snippet: Chimeric constructs, protein expression and putative oligomeric formations. ( A ) Representative cartoon of the fusion proteins. All chimeras were tagged with either YFP or CFP. White and black barrels represent Kv1.3 peptides. Dark and light gray correspond to KCNE4 structures. In KCNE4-Kv1.3 and KCNE4-Kv1.3T, the 18 aa link is also indicated. ( B ) Western blot of the protein lysates of the nontransfected HEK-293 cells and HEK-293 cells transfected with KCNE4 and Kv1.3. ( C ) Protein levels of cells expressing Kv1.3T, KCNE4-Kv1.3T and KCNE4-Kv1.3. ( D ) Putative oligomerization of Kv1.3 and KCNE4 complexes according to the construct combination. Basic channels formed by chimeras exhibited fixed stoichiometries. The addition of free KCNE4 units yielded forced channels with putative stoichiometries. 1–4, the number of KCNE units by complex, which varied from 1 to 4. 2–4, the number of KCNE units by complex, which varied from 2 to 4. White and black circles represent Kv1.3 peptides. Light gray corresponds to KCNE4 chimeras linked to Kv1.3. Dark gray highlights excess KCNE4 units.

Techniques Used: Construct, Expressing, Western Blot, Transfection

Current density versus voltage of K + currents. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and the K + currents were analyzed. Cells were clamped at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. ( A ) Representative traces from chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), and KCNE4:Kv1.3 (4:4). ( B ) Representative traces from functional complexes with putative Kv1.3-KCNE4 stoichiometries due to the addition of excess free KCNE4 units to Kv1.3-KCNE4 chimeras. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). ( C ) Current density (pA/pF) plotted against voltage (mV). ( D ) Peak current densities, at +60 mV, of different combinations without or with + KCNE4 and free KCNE4 added. Values are the means ± SE of 8–14 cells; ** p
Figure Legend Snippet: Current density versus voltage of K + currents. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and the K + currents were analyzed. Cells were clamped at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. ( A ) Representative traces from chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), and KCNE4:Kv1.3 (4:4). ( B ) Representative traces from functional complexes with putative Kv1.3-KCNE4 stoichiometries due to the addition of excess free KCNE4 units to Kv1.3-KCNE4 chimeras. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). ( C ) Current density (pA/pF) plotted against voltage (mV). ( D ) Peak current densities, at +60 mV, of different combinations without or with + KCNE4 and free KCNE4 added. Values are the means ± SE of 8–14 cells; ** p

Techniques Used: Transfection, Construct, Functional Assay

2) Product Images from "Imaging Kv1.3 Expressing Memory T Cells as a Marker of Immunotherapy Response"

Article Title: Imaging Kv1.3 Expressing Memory T Cells as a Marker of Immunotherapy Response

Journal: Cancers

doi: 10.3390/cancers14051217

Multicolour flow cytometry analysis of tumour-associated Kv1.3-expressing T cells after treatment. Percentages of ( A ) Kv1.3 CD4 + T EM cells relative to total CD4 + cells ( B ) CD4 + T EM cells relative to total CD4 + cells MFI ( C ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI ( D ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI between TR and TNR. Data are shown as individual values with the mean ± S.D. and are representative of n = 8–10 mice/ group. ** p
Figure Legend Snippet: Multicolour flow cytometry analysis of tumour-associated Kv1.3-expressing T cells after treatment. Percentages of ( A ) Kv1.3 CD4 + T EM cells relative to total CD4 + cells ( B ) CD4 + T EM cells relative to total CD4 + cells MFI ( C ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI ( D ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI between TR and TNR. Data are shown as individual values with the mean ± S.D. and are representative of n = 8–10 mice/ group. ** p

Techniques Used: Flow Cytometry, Expressing, Mouse Assay

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  • 93
    Millipore antibodies against kv1 3
    Analysis of <t>Kv1.3</t> activation kinetics. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (-) free KCNE4. Different combinations yielded fixed or putative Kv1.3:KCNE4 stoichiometries. Cells were held at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. Peak currents from −20 mV to +60 mV were calculated. ( A ) Time to reach the peak current in the open channels from −20 mV to +60 mV. The time scale is presented in log values for easier viewing. ( B ) Time to peak at 0 mV. Values are the means ± SE of 8–14 cells; * p
    Antibodies Against Kv1 3, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against kv1 3/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies against kv1 3 - by Bioz Stars, 2022-09
    93/100 stars
      Buy from Supplier

    95
    Millipore anti potassium channel kv1 3 extracellular antibody
    <t>Kv1.3</t> redistribution in the immunological synapse occurs by lateral movement of membrane channels. A. Kv1.3 (left) and CD3ε (right) distribution in resting T cells (not exposed to CD3/CD28 beads) in Kv1.3 crosslinking (XL) and control (CTR) cells.
    Anti Potassium Channel Kv1 3 Extracellular Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti potassium channel kv1 3 extracellular antibody/product/Millipore
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti potassium channel kv1 3 extracellular antibody - by Bioz Stars, 2022-09
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    99
    Millipore monoclonal antibody against kv1 1 α subunit
    Fig. 1. Nogo-A clusters at the paranodes in the CNS. ( A ) Adult rat brainstem sections were double immunolabeled for Nogo-A (green: a, c, d, f, g and i) and the <t>Kv1.1</t> K + channel <t>α-subunit</t> (red: b, c, h and i) or PAN Na + channel (red: e and f). For negative control, Nogo-A antiserum (1:200) was premixed with antigen before staining an adult brainstem section (j). Nogo-A antibodies used were those developed in our laboratory (a–f) or from Dr Strittmatter’s laboratory (g–i). Images c, f and i are merged images of a and b, d and e, and g and h, respectively. ( B ) Ultrastructural localization of Nogo-A at the paranodes in rat spinal cord: (a) immunogold labeling of cross-sections of myelinated axons revealed that the gold particles were detected at the inner and outer myelin sheaths; (b and c) immunogold particles of Nogo-A are found within glial loops and the compacted myelin; (d and e) in longitudinal sections of paranodes, immunogold particles of Nogo-A located at the tips of glial loops in the axoglial junction and some within the axon in (d). The boxed areas in (d) and (e) are shown at higher magnification in (d′) and (e′).OL, oligodendrocyte; ax, axon. Gold particles are indicated with arrows. Scale bars: 5 µm for (A, a–i), 10 µm for (A, j), 200 nm for (B, a, d and e) and 100 nm for (B, b, c, d′ and e′).
    Monoclonal Antibody Against Kv1 1 α Subunit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal antibody against kv1 1 α subunit/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monoclonal antibody against kv1 1 α subunit - by Bioz Stars, 2022-09
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    Image Search Results


    Analysis of Kv1.3 activation kinetics. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (-) free KCNE4. Different combinations yielded fixed or putative Kv1.3:KCNE4 stoichiometries. Cells were held at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. Peak currents from −20 mV to +60 mV were calculated. ( A ) Time to reach the peak current in the open channels from −20 mV to +60 mV. The time scale is presented in log values for easier viewing. ( B ) Time to peak at 0 mV. Values are the means ± SE of 8–14 cells; * p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Analysis of Kv1.3 activation kinetics. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (-) free KCNE4. Different combinations yielded fixed or putative Kv1.3:KCNE4 stoichiometries. Cells were held at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. Peak currents from −20 mV to +60 mV were calculated. ( A ) Time to reach the peak current in the open channels from −20 mV to +60 mV. The time scale is presented in log values for easier viewing. ( B ) Time to peak at 0 mV. Values are the means ± SE of 8–14 cells; * p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Activation Assay, Transfection

    KCNE4 specifically modulates Kv1.3. HEK-293 cells were transfected with Kv1.3-YFP in the presence (+KCNE4) or the absence of KCNE4-CFP. Cells were held at −80 mV, and currents were elicited by 1 s depolarizing pulses from −60 mV to +60 mV in 10 mV intervals. ( A ) Representative traces at +60 mV. ( B ) Current density vs. the voltage of K + currents ( p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: KCNE4 specifically modulates Kv1.3. HEK-293 cells were transfected with Kv1.3-YFP in the presence (+KCNE4) or the absence of KCNE4-CFP. Cells were held at −80 mV, and currents were elicited by 1 s depolarizing pulses from −60 mV to +60 mV in 10 mV intervals. ( A ) Representative traces at +60 mV. ( B ) Current density vs. the voltage of K + currents ( p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection

    GFP bleaching steps from HEK-293 cells transfected with Kv1.3 in the absence or presence of KCNE4. ( A – C ) Cells were transfected with Kv1.3-loopBAD-GFP. ( A ) Representative 1.5 s and 6.5 s time-lapse snapshots from the TIRFM video (488 nm laser). White arrow heads (a, b) point to the yellow square ROI (6 × 6 pixels) in panel c. (a) Initial fluorescence intensity; (b) fluorescence intensity after the first bleaching step at the same spot; (c) merge image. Yellow square indicates the ROI. The white line delineates the cell shape. Scale bar, 5 μm. ( B ) Representative graph of the bleaching steps for the different spots analyzed. Red arrows point to 4 bleaching steps. Squares, at the right, represent the fluorescence intensity after 4 consecutive bleaching steps at the same ROI. 0 s, initial fluorescence; 43.9 s, background intensity remaining after 4 consecutive bleaching steps. ( C ) Relative frequency of 1–4 bleaching events counted per spot. The black bars correspond to the experimental frequencies observed. Red dashed lines correspond to the theoretical distributions of bleaching steps with p = 0.67. ( D – G ) GFP bleaching steps from HEK-293 cells transfected with KCNE4-loopBAD-GFP and Kv1.3-Apple. ( D ) Representative snapshots from a cropped TIRF microscopy video. Upper panel (561 nm laser): Kv1.3-Apple. Lower panel (488 nm laser): KCNE4-loop-BADGFP. Merge panel zooms squares from previous panels. White squares ROIs (6 × 6 pixels) indicate colocalizing stationary spots. Putative oligomeric composition of the Kv1.3/KCNE4 complex is represented with circles at the left. While 4 white circles indicate the Kv1.3 tetramer, KCNE4 units are represented with gray and black circles. ( E – G ) Representative graph of the GFP bleaching steps from the different spots analyzed. ( E ) Two bleaching steps indicating a 4:2 Kv1.3:KCNE4 stoichiometry. ( F ) Three bleaching steps suggesting a 4:3 Kv1.3:KCNE4 ratio. ( G ) Four bleaching steps highlighting a 4:4 Kv1.3:KCNE4 stoichiometry. ( H ) Histogram with the relative frequency of 1–4 bleaching events counted for spot. Bars correspond to the experimental frequencies observed. ( I ) Histograms representing the theoretical distribution of the bleaching events when KCNE4 is present at the Kv1.3/KCNE4 complex as a monomer (4:1 stoichiometry), dimer (4:2), trimer (4:3) or tetramer (4:4) with p = 0.67. In contrast to the functional Kv1.3 tetramers ( C ), no expected KCNE4 distribution fit with the distribution observed during the analysis of the experimental data.

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: GFP bleaching steps from HEK-293 cells transfected with Kv1.3 in the absence or presence of KCNE4. ( A – C ) Cells were transfected with Kv1.3-loopBAD-GFP. ( A ) Representative 1.5 s and 6.5 s time-lapse snapshots from the TIRFM video (488 nm laser). White arrow heads (a, b) point to the yellow square ROI (6 × 6 pixels) in panel c. (a) Initial fluorescence intensity; (b) fluorescence intensity after the first bleaching step at the same spot; (c) merge image. Yellow square indicates the ROI. The white line delineates the cell shape. Scale bar, 5 μm. ( B ) Representative graph of the bleaching steps for the different spots analyzed. Red arrows point to 4 bleaching steps. Squares, at the right, represent the fluorescence intensity after 4 consecutive bleaching steps at the same ROI. 0 s, initial fluorescence; 43.9 s, background intensity remaining after 4 consecutive bleaching steps. ( C ) Relative frequency of 1–4 bleaching events counted per spot. The black bars correspond to the experimental frequencies observed. Red dashed lines correspond to the theoretical distributions of bleaching steps with p = 0.67. ( D – G ) GFP bleaching steps from HEK-293 cells transfected with KCNE4-loopBAD-GFP and Kv1.3-Apple. ( D ) Representative snapshots from a cropped TIRF microscopy video. Upper panel (561 nm laser): Kv1.3-Apple. Lower panel (488 nm laser): KCNE4-loop-BADGFP. Merge panel zooms squares from previous panels. White squares ROIs (6 × 6 pixels) indicate colocalizing stationary spots. Putative oligomeric composition of the Kv1.3/KCNE4 complex is represented with circles at the left. While 4 white circles indicate the Kv1.3 tetramer, KCNE4 units are represented with gray and black circles. ( E – G ) Representative graph of the GFP bleaching steps from the different spots analyzed. ( E ) Two bleaching steps indicating a 4:2 Kv1.3:KCNE4 stoichiometry. ( F ) Three bleaching steps suggesting a 4:3 Kv1.3:KCNE4 ratio. ( G ) Four bleaching steps highlighting a 4:4 Kv1.3:KCNE4 stoichiometry. ( H ) Histogram with the relative frequency of 1–4 bleaching events counted for spot. Bars correspond to the experimental frequencies observed. ( I ) Histograms representing the theoretical distribution of the bleaching events when KCNE4 is present at the Kv1.3/KCNE4 complex as a monomer (4:1 stoichiometry), dimer (4:2), trimer (4:3) or tetramer (4:4) with p = 0.67. In contrast to the functional Kv1.3 tetramers ( C ), no expected KCNE4 distribution fit with the distribution observed during the analysis of the experimental data.

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection, Fluorescence, Microscopy, Functional Assay

    Analysis of C-type inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (−) free KCNE4. Different combinations yielded fixed and putative Kv1.3:KCNE4 stoichiometries. ( A ) Representative traces from C-type inactivation recordings. An initial 100 ms pulse at −80 mV was applied prior to a 5 s depolarizing pulse at + 60 mV. ( B ) Relative intensity of voltage-dependent K + currents from all combinations during the first 2 s. ( C ) The constant (τ) of inactivation (in ms) of all groups. Values are the means ± SE, n = 8–14; * p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Analysis of C-type inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (−) free KCNE4. Different combinations yielded fixed and putative Kv1.3:KCNE4 stoichiometries. ( A ) Representative traces from C-type inactivation recordings. An initial 100 ms pulse at −80 mV was applied prior to a 5 s depolarizing pulse at + 60 mV. ( B ) Relative intensity of voltage-dependent K + currents from all combinations during the first 2 s. ( C ) The constant (τ) of inactivation (in ms) of all groups. Values are the means ± SE, n = 8–14; * p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection

    Analysis of the cumulative inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and K + currents were analyzed. ( A ) Chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), KCNE4:Kv1.3 (4:4). ( B ) Functional complexes with putative Kv1.3-KCNE4 stoichiometries due to further addition of extra free KCNE4 units. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). Cells were held at −80 mV, and a train of 25 depolarizing pulses to +60 mV for 200 ms was applied. ( C ) I/Imax vs. the pulse number. The ratio of the peak current amplitude during each pulse relative to that during the 1st pulse (I/Imax) was plotted against every pulse. ( D ) I/Imax reached at the last pulse train of depolarization. * p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Analysis of the cumulative inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and K + currents were analyzed. ( A ) Chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), KCNE4:Kv1.3 (4:4). ( B ) Functional complexes with putative Kv1.3-KCNE4 stoichiometries due to further addition of extra free KCNE4 units. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). Cells were held at −80 mV, and a train of 25 depolarizing pulses to +60 mV for 200 ms was applied. ( C ) I/Imax vs. the pulse number. The ratio of the peak current amplitude during each pulse relative to that during the 1st pulse (I/Imax) was plotted against every pulse. ( D ) I/Imax reached at the last pulse train of depolarization. * p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection, Construct, Functional Assay

    Chimeric constructs, protein expression and putative oligomeric formations. ( A ) Representative cartoon of the fusion proteins. All chimeras were tagged with either YFP or CFP. White and black barrels represent Kv1.3 peptides. Dark and light gray correspond to KCNE4 structures. In KCNE4-Kv1.3 and KCNE4-Kv1.3T, the 18 aa link is also indicated. ( B ) Western blot of the protein lysates of the nontransfected HEK-293 cells and HEK-293 cells transfected with KCNE4 and Kv1.3. ( C ) Protein levels of cells expressing Kv1.3T, KCNE4-Kv1.3T and KCNE4-Kv1.3. ( D ) Putative oligomerization of Kv1.3 and KCNE4 complexes according to the construct combination. Basic channels formed by chimeras exhibited fixed stoichiometries. The addition of free KCNE4 units yielded forced channels with putative stoichiometries. 1–4, the number of KCNE units by complex, which varied from 1 to 4. 2–4, the number of KCNE units by complex, which varied from 2 to 4. White and black circles represent Kv1.3 peptides. Light gray corresponds to KCNE4 chimeras linked to Kv1.3. Dark gray highlights excess KCNE4 units.

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Chimeric constructs, protein expression and putative oligomeric formations. ( A ) Representative cartoon of the fusion proteins. All chimeras were tagged with either YFP or CFP. White and black barrels represent Kv1.3 peptides. Dark and light gray correspond to KCNE4 structures. In KCNE4-Kv1.3 and KCNE4-Kv1.3T, the 18 aa link is also indicated. ( B ) Western blot of the protein lysates of the nontransfected HEK-293 cells and HEK-293 cells transfected with KCNE4 and Kv1.3. ( C ) Protein levels of cells expressing Kv1.3T, KCNE4-Kv1.3T and KCNE4-Kv1.3. ( D ) Putative oligomerization of Kv1.3 and KCNE4 complexes according to the construct combination. Basic channels formed by chimeras exhibited fixed stoichiometries. The addition of free KCNE4 units yielded forced channels with putative stoichiometries. 1–4, the number of KCNE units by complex, which varied from 1 to 4. 2–4, the number of KCNE units by complex, which varied from 2 to 4. White and black circles represent Kv1.3 peptides. Light gray corresponds to KCNE4 chimeras linked to Kv1.3. Dark gray highlights excess KCNE4 units.

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Construct, Expressing, Western Blot, Transfection

    Current density versus voltage of K + currents. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and the K + currents were analyzed. Cells were clamped at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. ( A ) Representative traces from chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), and KCNE4:Kv1.3 (4:4). ( B ) Representative traces from functional complexes with putative Kv1.3-KCNE4 stoichiometries due to the addition of excess free KCNE4 units to Kv1.3-KCNE4 chimeras. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). ( C ) Current density (pA/pF) plotted against voltage (mV). ( D ) Peak current densities, at +60 mV, of different combinations without or with + KCNE4 and free KCNE4 added. Values are the means ± SE of 8–14 cells; ** p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Current density versus voltage of K + currents. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and the K + currents were analyzed. Cells were clamped at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. ( A ) Representative traces from chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), and KCNE4:Kv1.3 (4:4). ( B ) Representative traces from functional complexes with putative Kv1.3-KCNE4 stoichiometries due to the addition of excess free KCNE4 units to Kv1.3-KCNE4 chimeras. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). ( C ) Current density (pA/pF) plotted against voltage (mV). ( D ) Peak current densities, at +60 mV, of different combinations without or with + KCNE4 and free KCNE4 added. Values are the means ± SE of 8–14 cells; ** p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection, Construct, Functional Assay

    Multicolour flow cytometry analysis of tumour-associated Kv1.3-expressing T cells after treatment. Percentages of ( A ) Kv1.3 CD4 + T EM cells relative to total CD4 + cells ( B ) CD4 + T EM cells relative to total CD4 + cells MFI ( C ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI ( D ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI between TR and TNR. Data are shown as individual values with the mean ± S.D. and are representative of n = 8–10 mice/ group. ** p

    Journal: Cancers

    Article Title: Imaging Kv1.3 Expressing Memory T Cells as a Marker of Immunotherapy Response

    doi: 10.3390/cancers14051217

    Figure Lengend Snippet: Multicolour flow cytometry analysis of tumour-associated Kv1.3-expressing T cells after treatment. Percentages of ( A ) Kv1.3 CD4 + T EM cells relative to total CD4 + cells ( B ) CD4 + T EM cells relative to total CD4 + cells MFI ( C ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI ( D ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI between TR and TNR. Data are shown as individual values with the mean ± S.D. and are representative of n = 8–10 mice/ group. ** p

    Article Snippet: Cells were stained with antibodies against Kv1.3 (polyclonal FITC; Sigma-Aldrich), CD103 (clone M290 FITC; BD Biosciences, San Jose, CA, USA), CD25 (clone PC61 BB700; BD Biosciences), CD45 (clone 30-F11 BUV395; BD Biosciences), Fixable Live/Dead Blue (Invitrogen, Waltham, MA, USA), CD62L (clone MEL-14 BUV563; BD Biosciences), CD86 (clone GL1 BUV615; BD Biosciences), F4/80 (clone T45-2342; BD Biosciences), NKp46 (clone 29A1.4 BUV737; BD Biosciences), CD3e (clone 500A2 BUV805; BD Biosciences), FoxP3 (clone 150D AlexaFluor647; Biolegend, San Diego, CA, USA), CD44 (clone IM7 APC-R700; BD Biosciences), CD11b (clone M1/70 APC-Cy7; Biolegend, San Diego, CA, USA), Granzyme B (clone QA16A02 PE; Biolegend), CCR7 (clone 4B12 PE-CF594; BD Biosciences), CD19 (clone 6D5 PE-Cy5; Biolegend), CD206 (clone C068C2 PE-Cy7; Biolegend), CD127 (clone SB/199 BV421; BD Biosciences), Ly6G (clone 1A8 BV480; BD Biosciences), CD8 (clone 53-6.7 BV510; BD Biosciences), CD11c (clone N418 BV570; Biolegend), Ly6C (clone HK1.4 BV605; Biolegend), Siglec F (clone E50-2440 BV650, BD Biosciences), CD68 (clone FA-11 BV711; Biolegend), CD4 (clone GK1.5 BV750; BD Biosciences), I-A/I-E (clone M5/114.15.2 BV785; Biolegend).

    Techniques: Flow Cytometry, Expressing, Mouse Assay

    Kv1.3 redistribution in the immunological synapse occurs by lateral movement of membrane channels. A. Kv1.3 (left) and CD3ε (right) distribution in resting T cells (not exposed to CD3/CD28 beads) in Kv1.3 crosslinking (XL) and control (CTR) cells.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Localization of Kv1.3 channels in the immunological synapse modulates the calcium response to antigen stimulation in T lymphocytes 1

    doi: 10.4049/jimmunol.0900613

    Figure Lengend Snippet: Kv1.3 redistribution in the immunological synapse occurs by lateral movement of membrane channels. A. Kv1.3 (left) and CD3ε (right) distribution in resting T cells (not exposed to CD3/CD28 beads) in Kv1.3 crosslinking (XL) and control (CTR) cells.

    Article Snippet: Briefly, Jurkat cells were incubated on ice for one hour with polyclonal anti-Kv1.3 antibody against an extracellular epitope (EC-Kv1.3, Sigma-Aldrich cat # P4497, St. Louis, MO) at 100:1 ratio of antibody:Kv1.3 α subunit (0.8 ug antibody/10 ml cell suspension of 2 million cells/ml), unless otherwise indicated.

    Techniques:

    Blockade of Kv1.3 movement to the immune synapse does not affect the distribution of Ca 2+ responses and the frequency of oscillations. A. Representative Ca 2+ responses induced by CD3/CD28 beads in individual cells. The points of introduction of the beads

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Localization of Kv1.3 channels in the immunological synapse modulates the calcium response to antigen stimulation in T lymphocytes 1

    doi: 10.4049/jimmunol.0900613

    Figure Lengend Snippet: Blockade of Kv1.3 movement to the immune synapse does not affect the distribution of Ca 2+ responses and the frequency of oscillations. A. Representative Ca 2+ responses induced by CD3/CD28 beads in individual cells. The points of introduction of the beads

    Article Snippet: Briefly, Jurkat cells were incubated on ice for one hour with polyclonal anti-Kv1.3 antibody against an extracellular epitope (EC-Kv1.3, Sigma-Aldrich cat # P4497, St. Louis, MO) at 100:1 ratio of antibody:Kv1.3 α subunit (0.8 ug antibody/10 ml cell suspension of 2 million cells/ml), unless otherwise indicated.

    Techniques:

    Specificity and lack of functional effects of extracellular anti-Kv1.3 antibody (EC Kv1.3 ab). A. Human T cells were fixed and stained with EC-Kv1.3 antibody (left) or anti-Kv1.3 antibody pre-adsorbed to the antigen (right). The corresponding DIC and

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Localization of Kv1.3 channels in the immunological synapse modulates the calcium response to antigen stimulation in T lymphocytes 1

    doi: 10.4049/jimmunol.0900613

    Figure Lengend Snippet: Specificity and lack of functional effects of extracellular anti-Kv1.3 antibody (EC Kv1.3 ab). A. Human T cells were fixed and stained with EC-Kv1.3 antibody (left) or anti-Kv1.3 antibody pre-adsorbed to the antigen (right). The corresponding DIC and

    Article Snippet: Briefly, Jurkat cells were incubated on ice for one hour with polyclonal anti-Kv1.3 antibody against an extracellular epitope (EC-Kv1.3, Sigma-Aldrich cat # P4497, St. Louis, MO) at 100:1 ratio of antibody:Kv1.3 α subunit (0.8 ug antibody/10 ml cell suspension of 2 million cells/ml), unless otherwise indicated.

    Techniques: Functional Assay, Staining

    Mature Kv1.3 channels move to the immunological synapse by lateral diffusion along the plane of the plasma membrane. A. Newly synthesized Kv1.3 channels are not recruited in the immune synapse. T cells were treated with cycloheximide (CHX, 10 uM) and

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Localization of Kv1.3 channels in the immunological synapse modulates the calcium response to antigen stimulation in T lymphocytes 1

    doi: 10.4049/jimmunol.0900613

    Figure Lengend Snippet: Mature Kv1.3 channels move to the immunological synapse by lateral diffusion along the plane of the plasma membrane. A. Newly synthesized Kv1.3 channels are not recruited in the immune synapse. T cells were treated with cycloheximide (CHX, 10 uM) and

    Article Snippet: Briefly, Jurkat cells were incubated on ice for one hour with polyclonal anti-Kv1.3 antibody against an extracellular epitope (EC-Kv1.3, Sigma-Aldrich cat # P4497, St. Louis, MO) at 100:1 ratio of antibody:Kv1.3 α subunit (0.8 ug antibody/10 ml cell suspension of 2 million cells/ml), unless otherwise indicated.

    Techniques: Diffusion-based Assay, Synthesized

    Blockade of Kv1.3 accumulation in the immune synapse alters Ca 2+ signaling. A. Ca 2+ response was measured by Fura-2 and elicited by CD3/CD28 beads in control (CTR) and Kv1.3 crosslinked (XL) cells. The average increase in [Ca 2+ ]i of responding cells is

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Localization of Kv1.3 channels in the immunological synapse modulates the calcium response to antigen stimulation in T lymphocytes 1

    doi: 10.4049/jimmunol.0900613

    Figure Lengend Snippet: Blockade of Kv1.3 accumulation in the immune synapse alters Ca 2+ signaling. A. Ca 2+ response was measured by Fura-2 and elicited by CD3/CD28 beads in control (CTR) and Kv1.3 crosslinked (XL) cells. The average increase in [Ca 2+ ]i of responding cells is

    Article Snippet: Briefly, Jurkat cells were incubated on ice for one hour with polyclonal anti-Kv1.3 antibody against an extracellular epitope (EC-Kv1.3, Sigma-Aldrich cat # P4497, St. Louis, MO) at 100:1 ratio of antibody:Kv1.3 α subunit (0.8 ug antibody/10 ml cell suspension of 2 million cells/ml), unless otherwise indicated.

    Techniques:

    Fig. 1. Nogo-A clusters at the paranodes in the CNS. ( A ) Adult rat brainstem sections were double immunolabeled for Nogo-A (green: a, c, d, f, g and i) and the Kv1.1 K + channel α-subunit (red: b, c, h and i) or PAN Na + channel (red: e and f). For negative control, Nogo-A antiserum (1:200) was premixed with antigen before staining an adult brainstem section (j). Nogo-A antibodies used were those developed in our laboratory (a–f) or from Dr Strittmatter’s laboratory (g–i). Images c, f and i are merged images of a and b, d and e, and g and h, respectively. ( B ) Ultrastructural localization of Nogo-A at the paranodes in rat spinal cord: (a) immunogold labeling of cross-sections of myelinated axons revealed that the gold particles were detected at the inner and outer myelin sheaths; (b and c) immunogold particles of Nogo-A are found within glial loops and the compacted myelin; (d and e) in longitudinal sections of paranodes, immunogold particles of Nogo-A located at the tips of glial loops in the axoglial junction and some within the axon in (d). The boxed areas in (d) and (e) are shown at higher magnification in (d′) and (e′).OL, oligodendrocyte; ax, axon. Gold particles are indicated with arrows. Scale bars: 5 µm for (A, a–i), 10 µm for (A, j), 200 nm for (B, a, d and e) and 100 nm for (B, b, c, d′ and e′).

    Journal: The EMBO Journal

    Article Title: Nogo-A at CNS paranodes is a ligand of Caspr: possible regulation of K+ channel localization

    doi: 10.1093/emboj/cdg570

    Figure Lengend Snippet: Fig. 1. Nogo-A clusters at the paranodes in the CNS. ( A ) Adult rat brainstem sections were double immunolabeled for Nogo-A (green: a, c, d, f, g and i) and the Kv1.1 K + channel α-subunit (red: b, c, h and i) or PAN Na + channel (red: e and f). For negative control, Nogo-A antiserum (1:200) was premixed with antigen before staining an adult brainstem section (j). Nogo-A antibodies used were those developed in our laboratory (a–f) or from Dr Strittmatter’s laboratory (g–i). Images c, f and i are merged images of a and b, d and e, and g and h, respectively. ( B ) Ultrastructural localization of Nogo-A at the paranodes in rat spinal cord: (a) immunogold labeling of cross-sections of myelinated axons revealed that the gold particles were detected at the inner and outer myelin sheaths; (b and c) immunogold particles of Nogo-A are found within glial loops and the compacted myelin; (d and e) in longitudinal sections of paranodes, immunogold particles of Nogo-A located at the tips of glial loops in the axoglial junction and some within the axon in (d). The boxed areas in (d) and (e) are shown at higher magnification in (d′) and (e′).OL, oligodendrocyte; ax, axon. Gold particles are indicated with arrows. Scale bars: 5 µm for (A, a–i), 10 µm for (A, j), 200 nm for (B, a, d and e) and 100 nm for (B, b, c, d′ and e′).

    Article Snippet: Kv1.1, Kv1.2 and Kv2.1 antibodies (Chemicon), monoclonal antibody against Kv1.1 α-subunit (K20/78), Kv1.2 α-subunit (K14/16, Upstate), Na+ channel (K58/35), NF-200 and MAP2 (Sigma) are from the respective commercial sources.

    Techniques: Immunolabeling, Negative Control, Staining, Labeling