Structured Review

NeuroMab kv1 1 antibody
Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic <t>Kv1.1</t> expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P
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1) Product Images from "Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1"

Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201212089

Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P
Figure Legend Snippet: Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P

Techniques Used: Over Expression, Expressing, Transfection

Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P
Figure Legend Snippet: Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P

Techniques Used: Over Expression, Infection, Expressing

HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P
Figure Legend Snippet: HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P

Techniques Used: Binding Assay, Western Blot, Isolation, Quantitative RT-PCR, Activated Clotting Time Assay

Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.
Figure Legend Snippet: Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.

Techniques Used: Sequencing, Binding Assay, In Situ Hybridization, Labeling

miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P
Figure Legend Snippet: miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P

Techniques Used: Sequencing, Binding Assay, Real-time Polymerase Chain Reaction

Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P
Figure Legend Snippet: Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P

Techniques Used: Sequencing, Imaging, Cell Culture, Expressing

mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P
Figure Legend Snippet: mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P

Techniques Used: Isolation, Western Blot

HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P
Figure Legend Snippet: HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P

Techniques Used: Expressing, Quantitative RT-PCR, Amplification, Northern Blot, Staining, Molecular Weight, Labeling, Reverse Transcription Polymerase Chain Reaction, Transfection, Binding Assay

2) Product Images from "A de novo KCNA1 Mutation in a Patient with Tetany and Hypomagnesemia"

Article Title: A de novo KCNA1 Mutation in a Patient with Tetany and Hypomagnesemia

Journal: Nephron. Clinical Practice

doi: 10.1159/000488954

Expression of wild-type and mutant Kv1.1 channels. a Cell surface biotinylation of HEK293 cells expressing either mock, Kv1.1 wild type, Kv1.1-p.Leu328Val, or co-expressing Kv1.1 wild type and Kv1.1-p.Leu328Val. Kv1.1 expression was analyzed by immunoblotting for plasma membrane fraction and input from the total cell lysates. Representative immunoblot of 4 independent experiments is shown. b Bar graph representing the quantification of the cell surface biotinylation experiments. It depicts the relative plasma membrane expression compared to input, and is shown as percentage of Kv1.1 wild type. Mean ± SEM is shown.
Figure Legend Snippet: Expression of wild-type and mutant Kv1.1 channels. a Cell surface biotinylation of HEK293 cells expressing either mock, Kv1.1 wild type, Kv1.1-p.Leu328Val, or co-expressing Kv1.1 wild type and Kv1.1-p.Leu328Val. Kv1.1 expression was analyzed by immunoblotting for plasma membrane fraction and input from the total cell lysates. Representative immunoblot of 4 independent experiments is shown. b Bar graph representing the quantification of the cell surface biotinylation experiments. It depicts the relative plasma membrane expression compared to input, and is shown as percentage of Kv1.1 wild type. Mean ± SEM is shown.

Techniques Used: Expressing, Mutagenesis

Electrophysiological analysis of Kv1.1 wild type and Kv1.1-p.Leu328Val channels. a Representative original traces of outward K + currents of HEK293 cells expressing mock, Kv1.1 wild type, or Kv1.1-p.Leu328Val. The insert on the right top shows the voltage protocol consisting of voltage steps from −100 to +50 mV in 10-mV increments, applied from a holding potential of −80 mV, every 10 s. b The current-voltage (I/V) relationships of mock (circles), Kv1.1 wild type (squares), Kv1.1-p.Leu328Val (triangles). Mean values are shown. c Histogram presenting the averaged current densities at +50 mV of mock ( n = 5), Kv1.1 wild type ( n = 12), Kv1.1-p.Leu328Val ( n = 6), and Kv1.1 wild type + Kv1.1-p.Leu328Val ( n = 9). Asterisk indicates p
Figure Legend Snippet: Electrophysiological analysis of Kv1.1 wild type and Kv1.1-p.Leu328Val channels. a Representative original traces of outward K + currents of HEK293 cells expressing mock, Kv1.1 wild type, or Kv1.1-p.Leu328Val. The insert on the right top shows the voltage protocol consisting of voltage steps from −100 to +50 mV in 10-mV increments, applied from a holding potential of −80 mV, every 10 s. b The current-voltage (I/V) relationships of mock (circles), Kv1.1 wild type (squares), Kv1.1-p.Leu328Val (triangles). Mean values are shown. c Histogram presenting the averaged current densities at +50 mV of mock ( n = 5), Kv1.1 wild type ( n = 12), Kv1.1-p.Leu328Val ( n = 6), and Kv1.1 wild type + Kv1.1-p.Leu328Val ( n = 9). Asterisk indicates p

Techniques Used: Expressing

3) Product Images from "Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1"

Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201212089

Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P
Figure Legend Snippet: Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P

Techniques Used: Over Expression, Expressing, Transfection

Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P
Figure Legend Snippet: Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P

Techniques Used: Over Expression, Infection, Expressing

HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P
Figure Legend Snippet: HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P

Techniques Used: Binding Assay, Western Blot, Isolation, Quantitative RT-PCR

Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.
Figure Legend Snippet: Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.

Techniques Used: Sequencing, Binding Assay, In Situ Hybridization, Labeling

miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P
Figure Legend Snippet: miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P

Techniques Used: Sequencing, Binding Assay, Real-time Polymerase Chain Reaction

Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P
Figure Legend Snippet: Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P

Techniques Used: Sequencing, Imaging, Cell Culture, Expressing

mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P
Figure Legend Snippet: mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P

Techniques Used: Isolation, Western Blot

HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P
Figure Legend Snippet: HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P

Techniques Used: Expressing, Quantitative RT-PCR, Amplification, Northern Blot, Staining, Molecular Weight, Labeling, Reverse Transcription Polymerase Chain Reaction, Transfection, Binding Assay

4) Product Images from "Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1"

Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201212089

Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P
Figure Legend Snippet: Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P

Techniques Used: Over Expression, Expressing, Transfection

Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P
Figure Legend Snippet: Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P

Techniques Used: Over Expression, Infection, Expressing

HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P
Figure Legend Snippet: HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P

Techniques Used: Binding Assay, Western Blot, Isolation, Quantitative RT-PCR, Activated Clotting Time Assay

Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.
Figure Legend Snippet: Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.

Techniques Used: Sequencing, Binding Assay, In Situ Hybridization, Labeling

miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P
Figure Legend Snippet: miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P

Techniques Used: Sequencing, Binding Assay, Real-time Polymerase Chain Reaction

Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P
Figure Legend Snippet: Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P

Techniques Used: Sequencing, Imaging, Cell Culture, Expressing

mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P
Figure Legend Snippet: mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P

Techniques Used: Isolation, Western Blot

HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P
Figure Legend Snippet: HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P

Techniques Used: Expressing, Quantitative RT-PCR, Amplification, Northern Blot, Staining, Molecular Weight, Labeling, Reverse Transcription Polymerase Chain Reaction, Transfection, Binding Assay

5) Product Images from "Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1"

Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201212089

Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P
Figure Legend Snippet: Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P

Techniques Used: Over Expression, Expressing, Transfection

Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P
Figure Legend Snippet: Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P

Techniques Used: Over Expression, Infection, Expressing

HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P
Figure Legend Snippet: HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P

Techniques Used: Binding Assay, Western Blot, Isolation, Quantitative RT-PCR

Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.
Figure Legend Snippet: Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.

Techniques Used: Sequencing, Binding Assay, In Situ Hybridization, Labeling

miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P
Figure Legend Snippet: miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P

Techniques Used: Sequencing, Binding Assay, Real-time Polymerase Chain Reaction

Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P
Figure Legend Snippet: Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P

Techniques Used: Sequencing, Imaging, Cell Culture, Expressing

mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P
Figure Legend Snippet: mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P

Techniques Used: Isolation, Western Blot

HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P
Figure Legend Snippet: HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P

Techniques Used: Expressing, Quantitative RT-PCR, Amplification, Northern Blot, Staining, Molecular Weight, Labeling, Reverse Transcription Polymerase Chain Reaction, Transfection, Binding Assay

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    Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic <t>Kv1.1</t> expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P
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    Quantitative evaluation of <t>Kv1.1</t> APs by the TopCorr-PV method. A , relative amounts of the core subunits of Kv1 channels as obtained in APs with the indicated anti-Kv1.1 Abs from CL-80 solubilized mouse brain membranes. The bars are the means ± S.D. of four to six specific peptide PVs normalized to their sum over all four APs; order of AP efficiency: anti-Kv1.1A (86.7%), anti-Kv1.1B (10.9%), and anti-Kv1.1C and <t>anti-Kv1.1D</t> (
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    Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P

    Journal: The Journal of Cell Biology

    Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

    doi: 10.1083/jcb.201212089

    Figure Lengend Snippet: Overexpression of the CaMKIIα UTR with multiple HuD sites occludes the increase in dendritic Kv1.1 expression. (A) Representative neurons (DIV 21) transfected with cDNA coding for EGFP or dGFP CaMKIIα UTRs were treated with 200 nM DMSO or rapamycin (Rapa), fixed, and immunostained for EGFP and Kv1.1. Quantification of dendritic Kv1.1 signal intensity for control (EGFP) or CaMKIIα UTR overexpression normalized by baseline signal for dendritic Kv1.1 under control conditions (EGFP/DMSO). Number of dendrites: DMSO-treated control, n = 22 and CaMKIIα UTR overexpression (OE), n = 29; rapamycin-treated control, n = 33 and CaMKIIα UTR overexpression, n = 31. *, P

    Article Snippet: For staining of surface-expressed Kv1.1, fixed but nonpermeabilized neurons were incubated overnight at 4°C with Kv1.1 antibody (1:1,000; NeuroMab) that recognizes the extracellular epitope.

    Techniques: Over Expression, Expressing, Transfection

    Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P

    Journal: The Journal of Cell Biology

    Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

    doi: 10.1083/jcb.201212089

    Figure Lengend Snippet: Overexpression of Kv1.1 3′UTR removes endogenous repression factors, leading to increased Kv1.1 protein. (left) Representative neurons infected with Sindbis virus coding for control RNA (Kaede-MAP2-DTS) or Kv1.1 3′UTR (DIV 21). Neurons were treated with DMSO or rapamycin. Arrowheads show Kv1.1 puncta (signal). Bar, 20 µm. (right) Quantification of surface expression of Kv1.1. ***, P

    Article Snippet: For staining of surface-expressed Kv1.1, fixed but nonpermeabilized neurons were incubated overnight at 4°C with Kv1.1 antibody (1:1,000; NeuroMab) that recognizes the extracellular epitope.

    Techniques: Over Expression, Infection, Expressing

    HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P

    Journal: The Journal of Cell Biology

    Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

    doi: 10.1083/jcb.201212089

    Figure Lengend Snippet: HuD binding to Kv1.1 mRNA coincides with reduced levels of other HuD-target mRNAs. (A) Western blot analysis of HuD from SNs isolated from neurons treated with DMSO or rapamycin. DMSO, n = 3; rapamycin (Rapa), n = 4 over two independent cultures. (B) RT-qPCR analysis of CaMKIIα, GAP-43, Homer1a, and Kv1.1 mRNA isolated from control or 200 nM rapamycin-treated neurons normalized to the internal housekeeping gene, GAPDH, which remains constant between the two conditions. CaMKIIα and Kv1.1, n = 3; GAP-43, n = 5; Homer1a, n = 4; three to five independent cultures. Control was normalized to 100% and is indicated by dotted line on the graph. The mRNA target of HuD is shown as percent remaining after rapamycin treatment. One-sample Student’s t test was performed to determine statistical significance from control. (C) Representative blot and quantification of SN CaMKIIα protein isolated from DMSO- or rapamycin-treated cortical neurons. DMSO, n = 5; rapamycin, n = 6. (D) Neurons were treated with 12 µM Actinomycin D (Act D) for 4–5 h before treating with DMSO or rapamycin for 75 min. Degradation was measured by RT-qPCR for CaMKIIα mRNA and reported as the percent decrease with the addition of rapamycin relative to actinomycin alone. n = 5 per treatment. The solid line is connecting the mean for actinomycin alone to the mean for 75- min DMSO + actinomycin treatment. The dotted line is connecting the mean for actinomycin alone to the mean for 75-min rapamycin + actinomycin treatment. *, P

    Article Snippet: For staining of surface-expressed Kv1.1, fixed but nonpermeabilized neurons were incubated overnight at 4°C with Kv1.1 antibody (1:1,000; NeuroMab) that recognizes the extracellular epitope.

    Techniques: Binding Assay, Western Blot, Isolation, Quantitative RT-PCR, Activated Clotting Time Assay

    Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

    doi: 10.1083/jcb.201212089

    Figure Lengend Snippet: Mutating the miR-129 seed match sequence in EGFP-Kv1.1 RNA increases protein levels without changing RNA levels. Representative images of dendritic localization of EGFP-Kv1.1 RNA (red) and protein (green) with the intact (left, FL) or mutated (right, ΔmiR-129) miR-129 binding site revealed by in situ hybridization (ISH) using a digoxigenin-labeled antisense oligo against EGFP. Bar, 20 µM. n = 12 and 13 neurons for FL and ΔmiR-129, 40 dendrites each. Error bars show SEMs.

    Article Snippet: For staining of surface-expressed Kv1.1, fixed but nonpermeabilized neurons were incubated overnight at 4°C with Kv1.1 antibody (1:1,000; NeuroMab) that recognizes the extracellular epitope.

    Techniques: Sequencing, Binding Assay, In Situ Hybridization, Labeling

    miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P

    Journal: The Journal of Cell Biology

    Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

    doi: 10.1083/jcb.201212089

    Figure Lengend Snippet: miR-129 binds Kv1.1 RNA when mTORC1 kinase is active. (A) Sequence alignment of Kv1.1 3′UTR indicates that the miR-129 seed match sequence (binding to nt 2–8 of miR-129) is conserved among rat, mouse, guinea pig, and human, with nt 1 and 8 being less conserved. The miR-129 binding site is highlighted in red. This motif is considered to be a “weak” binding site, consistent with a role in translational repression over degradation ( Vatolin et al., 2006 ; Grimson et al., 2007 ). Note that there are an additional 180 nt after the stop codon in the 3′UTR of the rat and mouse sequences that are not present in guinea pig and human sequences. Nucleotide number after the stop codon of each sequence shown: Rat, 181–212 nt; mouse, 177–207 nt; guinea pig, 1–34 nt; human, 1–35 nt. The NCBI accession numbers are rat M26161.1 , mouse NM_010595 , and human BC101733.1 . For guinea pig, the University of California, Santa Cruz genome browser database was used. The sequence is located in scaffold_107:2955798–2955831. The asterisks represent the nucleotides that are conserved between rat, mouse, guinea pig, and human. (B, top) Schematic of RNA fragments used as bait to determine miR-129 binding to Kv1.1. mTRS is indicated by the gray box illustrating the miR-129 seed match sequence (FL) or the mutated sequence (ΔmiR-129). (bottom) qPCR of miR-129 pulled down from DMSO- or rapamycin-treated neurons. *, P

    Article Snippet: For staining of surface-expressed Kv1.1, fixed but nonpermeabilized neurons were incubated overnight at 4°C with Kv1.1 antibody (1:1,000; NeuroMab) that recognizes the extracellular epitope.

    Techniques: Sequencing, Binding Assay, Real-time Polymerase Chain Reaction

    Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P

    Journal: The Journal of Cell Biology

    Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

    doi: 10.1083/jcb.201212089

    Figure Lengend Snippet: Mutating the miR-129 seed match sequence in Kaede-Kv1.1 mRNA results in mTORC1-independent new translation in neuronal dendrites. Live imaging of cultured hippocampal neurons expressing Kaede-Kv1.1 with (top, FL) or without (bottom, ΔmiR-129) seed match sequence of miR-129 in aCSF containing 200 nM DMSO (left, control) or rapamycin (right) before, immediately after (0 time point), and 120 min after UV exposure to photoconvert Kaede-Kv1.1. (left) Entire representative neuron. (right) Enlarged representative dendrite, indicated by arrows, > 60 µm from the soma. Bars: (main images) 50 µm; (insets) 10 µm. DMSO: FL, n = 58 puncta and ΔmiR129, n = 67 puncta; rapamycin (Rapa): FL, n = 51 puncta and ΔmiR-129, n = 64. *, P

    Article Snippet: For staining of surface-expressed Kv1.1, fixed but nonpermeabilized neurons were incubated overnight at 4°C with Kv1.1 antibody (1:1,000; NeuroMab) that recognizes the extracellular epitope.

    Techniques: Sequencing, Imaging, Cell Culture, Expressing

    mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P

    Journal: The Journal of Cell Biology

    Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

    doi: 10.1083/jcb.201212089

    Figure Lengend Snippet: mTORC1 kinase–dependent repression of Kv1.1 is not a result of mRNA stability. (A) SNs were isolated from DMSO- or rapamycin-treated DIV 21 cortical neurons. Representative Western blots and quantification indicate the relative level of p-mTOR/mTOR and Kv1.1/tubulin (loading control). ***, P

    Article Snippet: For staining of surface-expressed Kv1.1, fixed but nonpermeabilized neurons were incubated overnight at 4°C with Kv1.1 antibody (1:1,000; NeuroMab) that recognizes the extracellular epitope.

    Techniques: Isolation, Western Blot

    HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P

    Journal: The Journal of Cell Biology

    Article Title: Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

    doi: 10.1083/jcb.201212089

    Figure Lengend Snippet: HuD binds Kv1.1 mRNA when mTORC1 kinase is inhibited and increases Kv1.1 expression that is reversed with cycloheximide. (A, left) RT-qPCR amplification of miR-129 from neurons treated with DMSO or rapamycin (Rapa). n = 3 independent cultures, and each sample was performed in triplicate. (right) Northern blot analysis showed no significant difference in the expression level of miR-129 between neurons treated with DMSO or rapamycin. The signal intensity for miR-129 was normalized to the signal intensity for let-7a, which remained constant between DMSO- or rapamycin-treated neurons. As a loading control, ethidium bromide–stained low molecular weight RNA is shown in the bottom blot (labeled as EtBr). n.s., not significant. (B) RT-PCR amplification of Kv1.1 mRNA pulled down by HuD in HEK293T cells. HEK293T cells were cotransfected with myc-HuD and Kaede-Kv1.1 FL, CR, or ΔmiR-129. Antimyc-coated beads were used to pull down HuD bound to Kv1.1 mRNA outlined above. Kv1.1 FL without HuD or Kaede in place of Kv1.1 (bottom) were transfected as controls and show no binding to Kv1.1 mRNA or Kaede RNA alone. n = 2 independent cultures. (C, left) Representative neurons (DIV 14) cotransfected with EGFP and either pcDNA or HuD cDNA. Neurons were treated with 50 µM DMSO or cycloheximide, and Kv1.1 and Kv4.2 were detected with specific antibodies. Arrowheads show Kv1.1 puncta (signal). Bars, 20 µm. (right) Quantification of Kv1.1 and Kv4.2 punctal intensity in dendrites. Number of dendrites: DMSO-treated Kv1.1 pCDNA, n = 15 and HuD, n = 18; Kv4.2 pCDNA, n = 17 and HuD, n = 16. Cycloheximide (cyclo)-treated Kv1.1 HuD, n = 14 and Kv4.2 HuD, n = 17. ***, P

    Article Snippet: For staining of surface-expressed Kv1.1, fixed but nonpermeabilized neurons were incubated overnight at 4°C with Kv1.1 antibody (1:1,000; NeuroMab) that recognizes the extracellular epitope.

    Techniques: Expressing, Quantitative RT-PCR, Amplification, Northern Blot, Staining, Molecular Weight, Labeling, Reverse Transcription Polymerase Chain Reaction, Transfection, Binding Assay

    Expression of wild-type and mutant Kv1.1 channels. a Cell surface biotinylation of HEK293 cells expressing either mock, Kv1.1 wild type, Kv1.1-p.Leu328Val, or co-expressing Kv1.1 wild type and Kv1.1-p.Leu328Val. Kv1.1 expression was analyzed by immunoblotting for plasma membrane fraction and input from the total cell lysates. Representative immunoblot of 4 independent experiments is shown. b Bar graph representing the quantification of the cell surface biotinylation experiments. It depicts the relative plasma membrane expression compared to input, and is shown as percentage of Kv1.1 wild type. Mean ± SEM is shown.

    Journal: Nephron. Clinical Practice

    Article Title: A de novo KCNA1 Mutation in a Patient with Tetany and Hypomagnesemia

    doi: 10.1159/000488954

    Figure Lengend Snippet: Expression of wild-type and mutant Kv1.1 channels. a Cell surface biotinylation of HEK293 cells expressing either mock, Kv1.1 wild type, Kv1.1-p.Leu328Val, or co-expressing Kv1.1 wild type and Kv1.1-p.Leu328Val. Kv1.1 expression was analyzed by immunoblotting for plasma membrane fraction and input from the total cell lysates. Representative immunoblot of 4 independent experiments is shown. b Bar graph representing the quantification of the cell surface biotinylation experiments. It depicts the relative plasma membrane expression compared to input, and is shown as percentage of Kv1.1 wild type. Mean ± SEM is shown.

    Article Snippet: Kv1.1 expression was analyzed by immunoblot analysis for the input and the plasma membrane fraction using the Kv1.1 antibody (1: 3,000 Neuromab, Davis, CA, USA) and GFP antibody (1: 5,000, Sigma).

    Techniques: Expressing, Mutagenesis

    Electrophysiological analysis of Kv1.1 wild type and Kv1.1-p.Leu328Val channels. a Representative original traces of outward K + currents of HEK293 cells expressing mock, Kv1.1 wild type, or Kv1.1-p.Leu328Val. The insert on the right top shows the voltage protocol consisting of voltage steps from −100 to +50 mV in 10-mV increments, applied from a holding potential of −80 mV, every 10 s. b The current-voltage (I/V) relationships of mock (circles), Kv1.1 wild type (squares), Kv1.1-p.Leu328Val (triangles). Mean values are shown. c Histogram presenting the averaged current densities at +50 mV of mock ( n = 5), Kv1.1 wild type ( n = 12), Kv1.1-p.Leu328Val ( n = 6), and Kv1.1 wild type + Kv1.1-p.Leu328Val ( n = 9). Asterisk indicates p

    Journal: Nephron. Clinical Practice

    Article Title: A de novo KCNA1 Mutation in a Patient with Tetany and Hypomagnesemia

    doi: 10.1159/000488954

    Figure Lengend Snippet: Electrophysiological analysis of Kv1.1 wild type and Kv1.1-p.Leu328Val channels. a Representative original traces of outward K + currents of HEK293 cells expressing mock, Kv1.1 wild type, or Kv1.1-p.Leu328Val. The insert on the right top shows the voltage protocol consisting of voltage steps from −100 to +50 mV in 10-mV increments, applied from a holding potential of −80 mV, every 10 s. b The current-voltage (I/V) relationships of mock (circles), Kv1.1 wild type (squares), Kv1.1-p.Leu328Val (triangles). Mean values are shown. c Histogram presenting the averaged current densities at +50 mV of mock ( n = 5), Kv1.1 wild type ( n = 12), Kv1.1-p.Leu328Val ( n = 6), and Kv1.1 wild type + Kv1.1-p.Leu328Val ( n = 9). Asterisk indicates p

    Article Snippet: Kv1.1 expression was analyzed by immunoblot analysis for the input and the plasma membrane fraction using the Kv1.1 antibody (1: 3,000 Neuromab, Davis, CA, USA) and GFP antibody (1: 5,000, Sigma).

    Techniques: Expressing

    Quantitative evaluation of Kv1.1 APs by the TopCorr-PV method. A , relative amounts of the core subunits of Kv1 channels as obtained in APs with the indicated anti-Kv1.1 Abs from CL-80 solubilized mouse brain membranes. The bars are the means ± S.D. of four to six specific peptide PVs normalized to their sum over all four APs; order of AP efficiency: anti-Kv1.1A (86.7%), anti-Kv1.1B (10.9%), and anti-Kv1.1C and anti-Kv1.1D (

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Extending the Dynamic Range of Label-free Mass Spectrometric Quantification of Affinity Purifications *

    doi: 10.1074/mcp.M111.007955

    Figure Lengend Snippet: Quantitative evaluation of Kv1.1 APs by the TopCorr-PV method. A , relative amounts of the core subunits of Kv1 channels as obtained in APs with the indicated anti-Kv1.1 Abs from CL-80 solubilized mouse brain membranes. The bars are the means ± S.D. of four to six specific peptide PVs normalized to their sum over all four APs; order of AP efficiency: anti-Kv1.1A (86.7%), anti-Kv1.1B (10.9%), and anti-Kv1.1C and anti-Kv1.1D (

    Article Snippet: Knaus, Medical University of Innsbruck, Austria ( )), anti-Kv1.1B (rabbit polyclonal; Alomone Labs APC-009), anti-Kv1.1C (mouse monoclonal K36/15; Neuromab 73–105), and anti-Kv1.1D (mouse monoclonal K20/78; Neuromab 73–007); preimmune control IgG was from rabbit (Millipore/Upstate, catalog number 12-370).

    Techniques:

    Kv1.3 CBD less triggered no ER-stress. HEK-293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A ) Representative immunoblots showing the expression of ER-stress markers. Untransfected cells treated or not with 2 µM Thapsigargin (Tg) were used as positive and negative controls of ER-stress, respectively. Total protein extracts were separated by SDS-PAGE and immunoblotted for YFP (Kv1.3), GRP78 (Bip), XBP1, ATF-4, eIF2α, eIF2α pS51, and β-actin (loading control). ( B–E ) Relative expression (arbitrary units, A.U.) of different ER-stress markers. Data are the mean ± SE (n = 3–6). *p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Kv1.3 CBD less triggered no ER-stress. HEK-293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A ) Representative immunoblots showing the expression of ER-stress markers. Untransfected cells treated or not with 2 µM Thapsigargin (Tg) were used as positive and negative controls of ER-stress, respectively. Total protein extracts were separated by SDS-PAGE and immunoblotted for YFP (Kv1.3), GRP78 (Bip), XBP1, ATF-4, eIF2α, eIF2α pS51, and β-actin (loading control). ( B–E ) Relative expression (arbitrary units, A.U.) of different ER-stress markers. Data are the mean ± SE (n = 3–6). *p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Transfection, Western Blot, Expressing, SDS Page

    The integrity of the CBD domain is involved in the surface expression of Kv1.3. HEK 293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A–C ) Representative confocal images show colocalization of Kv1.3YFP WT and Kv1.3YFP CBD less with ( A ) plasma membrane (Mb), ( B ) Golgi, and ( C ) endoplasmic reticulum (ER). Green panels, Kv1.3; red panels, subcellular marker; merge panels show colocalization in yellow. The scale bar is 10 μm. ER (pDsRed-ER) and Mb (Akt-PH-pDsRed) were used as ER and Mb markers, respectively, and were cotransfected with the channel. Golgi was stained with an anti- cis -Golgi antibody (GM130). ( D ) Colocalization analysis (Pearson’s coefficient) between channel and subcellular markers. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Data are the mean ± SE (n > 30 cells) **p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: The integrity of the CBD domain is involved in the surface expression of Kv1.3. HEK 293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A–C ) Representative confocal images show colocalization of Kv1.3YFP WT and Kv1.3YFP CBD less with ( A ) plasma membrane (Mb), ( B ) Golgi, and ( C ) endoplasmic reticulum (ER). Green panels, Kv1.3; red panels, subcellular marker; merge panels show colocalization in yellow. The scale bar is 10 μm. ER (pDsRed-ER) and Mb (Akt-PH-pDsRed) were used as ER and Mb markers, respectively, and were cotransfected with the channel. Golgi was stained with an anti- cis -Golgi antibody (GM130). ( D ) Colocalization analysis (Pearson’s coefficient) between channel and subcellular markers. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Data are the mean ± SE (n > 30 cells) **p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Expressing, Transfection, Marker, Staining

    Caveolin modulates the pro-apoptotic activity of Kv1.3. ( A ) Subcellular fractionation was used to isolate mitochondrial (Mit) and plasma membrane (Mb) fractions in HEK-293 cells transfected with Kv1.3YFP WT. Samples were immunoblotted for GFP (Kv1.3), Caveolin, Na + /K + ATPase, and VDAC. ( B, C ) Electron micrographs showing HEK-293 cells transfected with Kv1.3YFP WT. Kv1.3 was immunolabeled with 18 nm gold particles (black arrowheads) and Cav1 with 12 nm gold particles (white arrowhead). The square inset in ( B ) indicates the zoomed in region in ( C ). Scale bars represent 500 nm. ( D ) Regular human Jurkat T lymphocytes express Kv1.3 and a negligible amount of endogenous Cav1 (Cav − ). In addition, a Jurkat cell line with notable expression of Cav1 was selected (Cav+). ( E ) Jurkat cells (Cav − and Cav + ) were electroporated with Kv1.3YFP WT or Kv1.3YFP CBD less . After 24 hr, apoptosis was assessed by Annexin V staining with flow cytometry. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBD less . ( F ) Mouse 3T3-L1 and 3T3-L1 Cav - preadipocytes were analyzed for the expression of endogenous Cav1 and Kv1.3. β-actin was used as a loading control. ( G ) Flow cytometric analysis quantifying apoptosis by Annexin V on 3T3-L1 (gray bar) and 3T3-L1 Cav - (white bar) preadipocytes. Note that the amount of Cav1 exerted notable effects on the Kv1.3-related apoptosis in native 3T3-L1 cells. Values are the mean ± SE of 3–6 independent experiments. *p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Caveolin modulates the pro-apoptotic activity of Kv1.3. ( A ) Subcellular fractionation was used to isolate mitochondrial (Mit) and plasma membrane (Mb) fractions in HEK-293 cells transfected with Kv1.3YFP WT. Samples were immunoblotted for GFP (Kv1.3), Caveolin, Na + /K + ATPase, and VDAC. ( B, C ) Electron micrographs showing HEK-293 cells transfected with Kv1.3YFP WT. Kv1.3 was immunolabeled with 18 nm gold particles (black arrowheads) and Cav1 with 12 nm gold particles (white arrowhead). The square inset in ( B ) indicates the zoomed in region in ( C ). Scale bars represent 500 nm. ( D ) Regular human Jurkat T lymphocytes express Kv1.3 and a negligible amount of endogenous Cav1 (Cav − ). In addition, a Jurkat cell line with notable expression of Cav1 was selected (Cav+). ( E ) Jurkat cells (Cav − and Cav + ) were electroporated with Kv1.3YFP WT or Kv1.3YFP CBD less . After 24 hr, apoptosis was assessed by Annexin V staining with flow cytometry. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBD less . ( F ) Mouse 3T3-L1 and 3T3-L1 Cav - preadipocytes were analyzed for the expression of endogenous Cav1 and Kv1.3. β-actin was used as a loading control. ( G ) Flow cytometric analysis quantifying apoptosis by Annexin V on 3T3-L1 (gray bar) and 3T3-L1 Cav - (white bar) preadipocytes. Note that the amount of Cav1 exerted notable effects on the Kv1.3-related apoptosis in native 3T3-L1 cells. Values are the mean ± SE of 3–6 independent experiments. *p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Activity Assay, Fractionation, Transfection, Immunolabeling, Expressing, Staining, Flow Cytometry

    Functional Kv1.3 CBD less channels exhibit altered biophysical properties and decreased current density at the plasma membrane. Kv1.3 WT and Kv1.3 CBD less were expressed in Xenopus oocytes to perform two-electrode voltage-clamp experiments. Cells were held at −100 mV and 2.5 s depolarizing pulses were applied in 20 mV steps to +40 mV. ( A ) Representative Kv1.3 protein expression of non-injected oocytes (control) and oocytes injected with Kv1.3WT (WT) and Kv1.3 CBD less (CBD less ). Representative currents elicited in a noninjected control oocyte ( B ) and either Kv1.3 WT ( C ) or Kv1.3 CBD less ( D ) expressing oocytes. ( E ) Peak current amplitude at +40 mV. ( F ) Time to peak at +40 mV. ( G ) Steady-state activation (left panel) and kinetic parameters (right panel). Data were fitted to a Boltzman equation. ( H ) Current inactivation at +40 mV and ( I ) τ inactivation values. Cumulative inactivation of Kv1.3 WT ( J ) and Kv1.3 CBD less ( K ) currents. Currents were elicited by a train of 8 depolarizing voltage steps to +40 mV once every second, from a holding potential of −100 mV. ( L ) Percentage of remaining current (I Kv1.3 ) at the end of the last pulse. ( M ) Membrane potential (Em (mV)) of non-injected (control) and injected oocytes with Kv1.3 WT or Kv1.3 CBD less . ( N ) Membrane input resistance (MΩ) of oocytes injected with Kv1.3 WT and Kv1.3 CBD less . Black bars, control (no injection); gray, oocytes injected with Kv1.3 WT; white bars, oocytes expressing Kv1.3 CBD less . Data are the mean ± SE (n: 20–26). *p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Functional Kv1.3 CBD less channels exhibit altered biophysical properties and decreased current density at the plasma membrane. Kv1.3 WT and Kv1.3 CBD less were expressed in Xenopus oocytes to perform two-electrode voltage-clamp experiments. Cells were held at −100 mV and 2.5 s depolarizing pulses were applied in 20 mV steps to +40 mV. ( A ) Representative Kv1.3 protein expression of non-injected oocytes (control) and oocytes injected with Kv1.3WT (WT) and Kv1.3 CBD less (CBD less ). Representative currents elicited in a noninjected control oocyte ( B ) and either Kv1.3 WT ( C ) or Kv1.3 CBD less ( D ) expressing oocytes. ( E ) Peak current amplitude at +40 mV. ( F ) Time to peak at +40 mV. ( G ) Steady-state activation (left panel) and kinetic parameters (right panel). Data were fitted to a Boltzman equation. ( H ) Current inactivation at +40 mV and ( I ) τ inactivation values. Cumulative inactivation of Kv1.3 WT ( J ) and Kv1.3 CBD less ( K ) currents. Currents were elicited by a train of 8 depolarizing voltage steps to +40 mV once every second, from a holding potential of −100 mV. ( L ) Percentage of remaining current (I Kv1.3 ) at the end of the last pulse. ( M ) Membrane potential (Em (mV)) of non-injected (control) and injected oocytes with Kv1.3 WT or Kv1.3 CBD less . ( N ) Membrane input resistance (MΩ) of oocytes injected with Kv1.3 WT and Kv1.3 CBD less . Black bars, control (no injection); gray, oocytes injected with Kv1.3 WT; white bars, oocytes expressing Kv1.3 CBD less . Data are the mean ± SE (n: 20–26). *p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Functional Assay, Expressing, Injection, Activation Assay

    Caveolin-1 protects from apoptosis when associated with Kv1.3 in primary human T lymphocytes. Human CD4+ T lymphocytes were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less and the mitochondrial membrane potential (TMRM) and apoptosis were measured. YFP-transfected cells were used as a control. ( A ) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Cells were incubated with TMRM and analyzed by flow cytometry. A.U, arbitrary units. ( B ) T cells were electroporated with Kv1.3YFP WT or Kv1.3YFP CBD less with (Cav+) or without (Cav−) Cav1 Cerulean. After 24 hr, transfected cells were sorted and apoptosis was assessed by Annexin V staining with flow cytometry. The level of apoptosis in arbitrary units (A.U.) was measured in each group by resting the value of basal apoptosis in cells transfected with YFP in the presence (Cav−) or the absence (Cav−) of Cav1. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBD less . Cav−, regular CD4+ cells without Cav 1; Cav+, T cells transfected with Cav1 Cer. Data are the mean ± SE (n = 5–7). *p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Caveolin-1 protects from apoptosis when associated with Kv1.3 in primary human T lymphocytes. Human CD4+ T lymphocytes were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less and the mitochondrial membrane potential (TMRM) and apoptosis were measured. YFP-transfected cells were used as a control. ( A ) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Cells were incubated with TMRM and analyzed by flow cytometry. A.U, arbitrary units. ( B ) T cells were electroporated with Kv1.3YFP WT or Kv1.3YFP CBD less with (Cav+) or without (Cav−) Cav1 Cerulean. After 24 hr, transfected cells were sorted and apoptosis was assessed by Annexin V staining with flow cytometry. The level of apoptosis in arbitrary units (A.U.) was measured in each group by resting the value of basal apoptosis in cells transfected with YFP in the presence (Cav−) or the absence (Cav−) of Cav1. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBD less . Cav−, regular CD4+ cells without Cav 1; Cav+, T cells transfected with Cav1 Cer. Data are the mean ± SE (n = 5–7). *p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Transfection, Fluorescence, Incubation, Flow Cytometry, Staining

    Flow cytometry evaluates apoptosis by Annexin V staining. Mouse melanoma B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less and treated for an additional 24 hr with different pro-apoptotic agents. Transfected cells were sorted and % of Annexin V-positive cells was obtained. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Data are the mean ± SE (n = 3). *p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Flow cytometry evaluates apoptosis by Annexin V staining. Mouse melanoma B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less and treated for an additional 24 hr with different pro-apoptotic agents. Transfected cells were sorted and % of Annexin V-positive cells was obtained. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Data are the mean ± SE (n = 3). *p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Flow Cytometry, Staining, Transfection

    Kv1.3 CBD less severely impairs mitochondrial function. HEK-293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . Non-transfected cells were used as a control. ( A ) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Positive transfected cells (separated by sorting) were incubated with TMRM and analyzed by confocal microscopy. Data are the mean ± SE (n = 3). ***p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Kv1.3 CBD less severely impairs mitochondrial function. HEK-293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . Non-transfected cells were used as a control. ( A ) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Positive transfected cells (separated by sorting) were incubated with TMRM and analyzed by confocal microscopy. Data are the mean ± SE (n = 3). ***p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Transfection, Fluorescence, Incubation, Confocal Microscopy

    Kv1.3 CBD less targets mitochondria altering mitochondrial morphology. HEK 293 and B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less . ( A ) Representative confocal images of HEK-293 cells cotransfected with (Aa–Ad) Kv1.3YFP WT, (Ae–Ah) Kv1.3YFP CBD less (green) and pmitoRFP (red). Images were processed (tubeness (Ac, Ag) and skeleton (Ad, Ah)) to perform morphometric analysis ( B–D ) of mitochondria in Kv1.3 positive cells. Scale bar represents 10 μm. ( B ) The form factor (arbitrary units, A.U.) describes the particle shape complexity and is computed as the average (perimeter) 2 /(4π·area). A circle corresponds to a minimum value of 1. ( C ) Average area of particles detected on the binary image. ( D ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. Data are the mean ± SE (n > 30). ***p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Kv1.3 CBD less targets mitochondria altering mitochondrial morphology. HEK 293 and B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less . ( A ) Representative confocal images of HEK-293 cells cotransfected with (Aa–Ad) Kv1.3YFP WT, (Ae–Ah) Kv1.3YFP CBD less (green) and pmitoRFP (red). Images were processed (tubeness (Ac, Ag) and skeleton (Ad, Ah)) to perform morphometric analysis ( B–D ) of mitochondria in Kv1.3 positive cells. Scale bar represents 10 μm. ( B ) The form factor (arbitrary units, A.U.) describes the particle shape complexity and is computed as the average (perimeter) 2 /(4π·area). A circle corresponds to a minimum value of 1. ( C ) Average area of particles detected on the binary image. ( D ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. Data are the mean ± SE (n > 30). ***p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Transfection

    Kv1.3 CBD less does not localize to the plasma membrane in B16F10 melanoma cells. B16F10 melanoma cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A ) Colocalization of Kv1.3YFP WT (Aa-Ac) and Kv1.3YFP CBD less (Ad-Af) at the plasma membrane. (Aa, Ad) Kv1.3YFP in green. (Ab, Ae) Plasma membrane staining with FM-464 dye in red. (Ac, Af) Merge shows colocalization in yellow. The scale bar represents 10 μm. ( B ) Quantification of colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n = 18). *p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Kv1.3 CBD less does not localize to the plasma membrane in B16F10 melanoma cells. B16F10 melanoma cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A ) Colocalization of Kv1.3YFP WT (Aa-Ac) and Kv1.3YFP CBD less (Ad-Af) at the plasma membrane. (Aa, Ad) Kv1.3YFP in green. (Ab, Ae) Plasma membrane staining with FM-464 dye in red. (Ac, Af) Merge shows colocalization in yellow. The scale bar represents 10 μm. ( B ) Quantification of colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n = 18). *p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Transfection, Staining

    Disruption of the CBD of Kv1.3 impairs caveolin colocalization, as well as association with the channel. HEK 293 Cav − cells were cotransfected with Kv1.3YFP WT and Kv1.3YFP mutants (F166A, W171G/F174A, and CBD less ) and Cav1. ( A ) Representative confocal images from an experiment on cell unroofing preparations (CUPs). HEK 293 Cav − cells were cotransfected with Kv1.3YFP WT+Cav1 or Kv1.3YFP mutants (F166A, W171G/F174A, and CBD less )+Cav1. (Aa, Ae, Ai) Kv1.3YFP WT; (Ab, Af, Aj) Kv1.3YFP (F166A); (Ac, Ag, Ak) Kv1.3YFP (W171G/F174A); (Ad, Ah, Al) Kv1.3YFP CBD less . Green, Kv1.3 channels; Red, Cav1; Merge, colocalization between Kv1.3 and Cav1 in yellow. The scale bar represents 10 μm. ( B ) Consensus sequence of the CBD. The amino acid sequence shows the CBD of wild type (WT) Kv1.3. The Kv1.3 CBD mutants contain amino acid substitutions (in red) to disrupt the CBD. ( C ) Pixel by pixel colocalization analysis of Kv1.3 channels and Cav1. Dark gray bar, Kv1.3 WT; light gray bar, Kv1.3 F166A mutant; black bar, Kv1.3 W171G/F174A; white bar, Kv1.3 CBD less . Values are the mean ± SE (n > 25). **p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Disruption of the CBD of Kv1.3 impairs caveolin colocalization, as well as association with the channel. HEK 293 Cav − cells were cotransfected with Kv1.3YFP WT and Kv1.3YFP mutants (F166A, W171G/F174A, and CBD less ) and Cav1. ( A ) Representative confocal images from an experiment on cell unroofing preparations (CUPs). HEK 293 Cav − cells were cotransfected with Kv1.3YFP WT+Cav1 or Kv1.3YFP mutants (F166A, W171G/F174A, and CBD less )+Cav1. (Aa, Ae, Ai) Kv1.3YFP WT; (Ab, Af, Aj) Kv1.3YFP (F166A); (Ac, Ag, Ak) Kv1.3YFP (W171G/F174A); (Ad, Ah, Al) Kv1.3YFP CBD less . Green, Kv1.3 channels; Red, Cav1; Merge, colocalization between Kv1.3 and Cav1 in yellow. The scale bar represents 10 μm. ( B ) Consensus sequence of the CBD. The amino acid sequence shows the CBD of wild type (WT) Kv1.3. The Kv1.3 CBD mutants contain amino acid substitutions (in red) to disrupt the CBD. ( C ) Pixel by pixel colocalization analysis of Kv1.3 channels and Cav1. Dark gray bar, Kv1.3 WT; light gray bar, Kv1.3 F166A mutant; black bar, Kv1.3 W171G/F174A; white bar, Kv1.3 CBD less . Values are the mean ± SE (n > 25). **p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Sequencing, Mutagenesis

    The accumulation of Kv1.3 in mitochondria is not responsible for the increase in apoptosis. HEK 293 cells were transfected with Kv1.3 WT, Kv1.3 CBD less , or the Kv1.3 YMVIii mutant. See Materials and methods for details. The pmitoRFP mitochondrial marker was also cotransfected to stain mitochondria. ( A ) Representative confocal images from WT (Aa–Ac), CBD less (Ad–Af), and YMVIii (Ag–Ai) channels. Green, Kv1.3 channels; red, mitochondria; merge, colocalization of channel and mitochondria in yellow. Bars represent 20 μm. ( B ) Quantification of Kv1.3/mitochondria colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 20 cells). ( C ) Coimmunoprecipitation of Kv1.3 channels and Cav1. HEK 293 lysates were immunoprecipitated against Cav1 (IP: Cav1) and blots were immunoblotted against YFP-tagged channels (IB: Kv1.3) and Cav1 (IB: Cav1). IP−, absence of anti-Cav1 antibody. SM, starting material. ( D ) Kv1.3-transfected cells were sorted and % of Annexin V-positive cells was obtained. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Black bars, Kv1.3 YMVIii. Data are the mean ± SE (n > 4). *p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: The accumulation of Kv1.3 in mitochondria is not responsible for the increase in apoptosis. HEK 293 cells were transfected with Kv1.3 WT, Kv1.3 CBD less , or the Kv1.3 YMVIii mutant. See Materials and methods for details. The pmitoRFP mitochondrial marker was also cotransfected to stain mitochondria. ( A ) Representative confocal images from WT (Aa–Ac), CBD less (Ad–Af), and YMVIii (Ag–Ai) channels. Green, Kv1.3 channels; red, mitochondria; merge, colocalization of channel and mitochondria in yellow. Bars represent 20 μm. ( B ) Quantification of Kv1.3/mitochondria colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 20 cells). ( C ) Coimmunoprecipitation of Kv1.3 channels and Cav1. HEK 293 lysates were immunoprecipitated against Cav1 (IP: Cav1) and blots were immunoblotted against YFP-tagged channels (IB: Kv1.3) and Cav1 (IB: Cav1). IP−, absence of anti-Cav1 antibody. SM, starting material. ( D ) Kv1.3-transfected cells were sorted and % of Annexin V-positive cells was obtained. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Black bars, Kv1.3 YMVIii. Data are the mean ± SE (n > 4). *p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Transfection, Mutagenesis, Marker, Staining, Immunoprecipitation

    Kv1.3 colocalizes with plasma membrane and mitochondria in primary human T lymphocytes. CD4+ lymphocytes were isolated from human blood as indicated in Materials and methods. ( A ) Representative western blot from HEK 293 cells and T lymphocytes samples from two independent human donors showing differential protein expression of Kv1.3, Flotillin, and Cav1. β-Actin was a loading control. ( B ) Representative confocal images of Kv1.3 colocalization in plasma membrane (Mb) from Kv1.3YFP WT and Kv1.3YFP CBD less -transfected cells. WGA stained plasma membrane. ( C ) Representative confocal images of Kv1.3 colocalization in mitochondria (mito) from Kv1.3YFP WT and Kv1.3YFP CBD less expressing cells. MitoTracker was used for mitochondrial staining. Scale bar represents 10 μm. Quantification of Kv1.3/Mb ( D ) and Kv1.3/mito ( E ) colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 20), Student’s t-test. Gray bars, Kv1.3YFP WT cells; white bars, Kv1.3YFP CBD less cells.

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Kv1.3 colocalizes with plasma membrane and mitochondria in primary human T lymphocytes. CD4+ lymphocytes were isolated from human blood as indicated in Materials and methods. ( A ) Representative western blot from HEK 293 cells and T lymphocytes samples from two independent human donors showing differential protein expression of Kv1.3, Flotillin, and Cav1. β-Actin was a loading control. ( B ) Representative confocal images of Kv1.3 colocalization in plasma membrane (Mb) from Kv1.3YFP WT and Kv1.3YFP CBD less -transfected cells. WGA stained plasma membrane. ( C ) Representative confocal images of Kv1.3 colocalization in mitochondria (mito) from Kv1.3YFP WT and Kv1.3YFP CBD less expressing cells. MitoTracker was used for mitochondrial staining. Scale bar represents 10 μm. Quantification of Kv1.3/Mb ( D ) and Kv1.3/mito ( E ) colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 20), Student’s t-test. Gray bars, Kv1.3YFP WT cells; white bars, Kv1.3YFP CBD less cells.

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Isolation, Western Blot, Expressing, Transfection, Whole Genome Amplification, Staining

    Kv1.3 CBD less forms tetramers. ( A ) Representative FRET experiment of CUPs from HEK 293 cells cotransfected with Kv1.3YFP WT+Kv1.3 Cer WT (left panels) or Kv1.3YFP CBD less +Kv1.3 Cer CBD less (right panels). Top to bottom: acceptor (Kv1.3-YFP) and donor (Kv1.3-Cer) prebleach and postbleach images. Square insets indicate the bleached zone, and the red area indicates the analyzed ROI. Line graphs show changes in donor (cerulean) and acceptor (yellow) fluorescence after bleaching. The scale bar represents 10 μm. ( B ) FRET efficiency (%) of YFP +Cer (negative control), Kv1.3YFP WT +Kv1.3 Cer WT (positive control), and Kv1.3YFP CBD less +Kv1.3 Cer CBD less . Values are mean ± SE (n > 20). ***p

    Journal: eLife

    Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

    doi: 10.7554/eLife.69099

    Figure Lengend Snippet: Kv1.3 CBD less forms tetramers. ( A ) Representative FRET experiment of CUPs from HEK 293 cells cotransfected with Kv1.3YFP WT+Kv1.3 Cer WT (left panels) or Kv1.3YFP CBD less +Kv1.3 Cer CBD less (right panels). Top to bottom: acceptor (Kv1.3-YFP) and donor (Kv1.3-Cer) prebleach and postbleach images. Square insets indicate the bleached zone, and the red area indicates the analyzed ROI. Line graphs show changes in donor (cerulean) and acceptor (yellow) fluorescence after bleaching. The scale bar represents 10 μm. ( B ) FRET efficiency (%) of YFP +Cer (negative control), Kv1.3YFP WT +Kv1.3 Cer WT (positive control), and Kv1.3YFP CBD less +Kv1.3 Cer CBD less . Values are mean ± SE (n > 20). ***p

    Article Snippet: The filters were then immunoblotted with specific antibodies: anti-GFP (1:500, Roche), anti-caveolin (1:250, BD Biosciences), anti-Kv1.3 (1:200, Neuromab), anti-clathrin (1:1000, BD Biosciences), anti-flotillin (1:500, BD Biosciences), anti-β actin (1:50,000, Sigma), anti-Na+ /K+ ATPase (Developmental Studies Hybridoma Bank, The University of Iowa), anti-VDAC (1:5000, Calbiochem), anti-GRP78 (1:1000, Cell Signaling Technology), anti-XBP1 (1:1000, Abcam), anti-ATF4 (1:500, Santa Cruz Biotechnologies), anti-eIF2α (1:1000, Abcam), and anti-eIF2α pS51 (1:1000, Abcam).

    Techniques: Fluorescence, Negative Control, Positive Control