Journal: American Journal of Physiology - Renal Physiology

Article Title: Furosemide reduces BK-αβ4-mediated K + secretion in mice on an alkaline high-K + diet

doi: 10.1152/ajprenal.00223.2018

Figure Lengend Snippet: Furosemide (Furo) effect on urine pH. Urine pH of wild-type (WT; A) and β4-knockout (β4-KO; B) on day −1, 1, 4, 7, and 11 of control (Ctrl) and Furo treatments or on day −1, 1, 4, and 7 of Furo + acetazolamide (Actz) treatment. *P < 0.05 vs. HK + Ctrl water, #P < 0.05 vs. Day –1, analyzed with two-way repeated-measures ANOVA with a post hoc Holm-Sidak test (P < 0.001 for both treatment and days with interaction P < 0.01). n = 12–14 for WT Ctrl and Furo; N = 7 – 8 for β4-KO Ctrl and Furo; N = 4 for WT Furo + Actz; n = 3 for β4-KO Furo + Actz.

Article Snippet: Primary antibodies included anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories, Jerusalem, Israel), anti-NHE3 (mouse monoclonal, diluted 1:200; Invitrogen, Carlsbad, CA), and anti-V-ATPase B1 (rabbit polyclonal, diluted 1:200; GeneTex, Irvine, CA).

Techniques: Knock-Out

Journal: American Journal of Physiology - Renal Physiology

Article Title: Furosemide reduces BK-αβ4-mediated K + secretion in mice on an alkaline high-K + diet

doi: 10.1152/ajprenal.00223.2018

Figure Lengend Snippet: Blood measurements after 11 days (or 7 days for HK + Furo Actz) of treatment

Article Snippet: Primary antibodies included anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories, Jerusalem, Israel), anti-NHE3 (mouse monoclonal, diluted 1:200; Invitrogen, Carlsbad, CA), and anti-V-ATPase B1 (rabbit polyclonal, diluted 1:200; GeneTex, Irvine, CA).

Techniques:

Journal: American Journal of Physiology - Renal Physiology

Article Title: Furosemide reduces BK-αβ4-mediated K + secretion in mice on an alkaline high-K + diet

doi: 10.1152/ajprenal.00223.2018

Figure Lengend Snippet: Furosemide (Furo) effect on plasma [K+] and renal K+ clearance in mice on regular diet (RD) and high-K+ diet (HK). A and B: plasma [K+] and renal K+ clearance of WT on RD on day 11 of control (Ctrl) or Furo treatments. *P < 0.05 vs. RD + Ctrl analyzed with Student’s t-test; n = 4 for Ctrl, n = 6 for Furo. C and D: plasma [K+] and renal K+ clearance of WT and β4-knockout (β4-KO) on day 11 of Ctrl and Furo treatment or day 7 of Furo + acetazolamide (Actz) treatment. *P < 0.05 vs. HK + Ctrl; #P < 0.05 vs. HK + Furo, analyzed with two-way ANOVA with a post hoc Tukey test (P < 0.001 for treatment in plasma [K+]; P < 0.01 for treatment in renal K+ clearance); n = 12 – 14 for WT HK + Ctrl and HK + Furo; n = 7 – 8 for β4-KO HK + Ctrl and HK + Furo; n = 4 for WT HK + Furo Actz; n = 3 for β4-KO HK + Furo Actz.

Article Snippet: Primary antibodies included anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories, Jerusalem, Israel), anti-NHE3 (mouse monoclonal, diluted 1:200; Invitrogen, Carlsbad, CA), and anti-V-ATPase B1 (rabbit polyclonal, diluted 1:200; GeneTex, Irvine, CA).

Techniques: Knock-Out

Journal: American Journal of Physiology - Renal Physiology

Article Title: Furosemide reduces BK-αβ4-mediated K + secretion in mice on an alkaline high-K + diet

doi: 10.1152/ajprenal.00223.2018

Figure Lengend Snippet: Furosemide (Furo) effect on urinary K+ excretion (UKV̇) normalized to kidney weight in mice on high-K diet (HK). UKV̇ of wild-type (WT; A) and β4-knockout (β4-KO; B) on day −1, 1, 4, 7, and 11 of control (Ctrl) and Furo treatment or on day −1, 1, 4, and 7 of Furo + acetazolamide (Actz) treatment. *P < 0.05 vs. HK + Ctrl; #P < 0.05 vs. day −1, analyzed with two-way repeated measures ANOVA (P < 0.05 for drug treatment in WT) with a post hoc Holm-Sidak test; n = 12–14 for WT HK + Ctrl and HK + Furo; n = 7–8 for β4-KO HK + Ctrl and HK + Furo; n = 4 for WT HK + Furo Actz; n = 3 for β4-KO HK + Furo Actz.

Article Snippet: Primary antibodies included anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories, Jerusalem, Israel), anti-NHE3 (mouse monoclonal, diluted 1:200; Invitrogen, Carlsbad, CA), and anti-V-ATPase B1 (rabbit polyclonal, diluted 1:200; GeneTex, Irvine, CA).

Techniques: Knock-Out

Journal: American Journal of Physiology - Renal Physiology

Article Title: Furosemide reduces BK-αβ4-mediated K + secretion in mice on an alkaline high-K + diet

doi: 10.1152/ajprenal.00223.2018

Figure Lengend Snippet: Metabolic cage measurements after 11 days (or 7 days for HK + Furo Actz) of treatment

Article Snippet: Primary antibodies included anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories, Jerusalem, Israel), anti-NHE3 (mouse monoclonal, diluted 1:200; Invitrogen, Carlsbad, CA), and anti-V-ATPase B1 (rabbit polyclonal, diluted 1:200; GeneTex, Irvine, CA).

Techniques:

Journal: American Journal of Physiology - Renal Physiology

Article Title: Furosemide reduces BK-αβ4-mediated K + secretion in mice on an alkaline high-K + diet

doi: 10.1152/ajprenal.00223.2018

Figure Lengend Snippet: Correlation between renal K+ clearance and urine pH in wild-type (WT; A) and β4-knockout (β4-KO; B) on high-K+ diet (HK). ●, HK + control (Ctrl) group; ○, HK + furosemide (Furo) group; ▲, HK + Furo acetazolamide (Actz) group; △, HKCl group.

Article Snippet: Primary antibodies included anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories, Jerusalem, Israel), anti-NHE3 (mouse monoclonal, diluted 1:200; Invitrogen, Carlsbad, CA), and anti-V-ATPase B1 (rabbit polyclonal, diluted 1:200; GeneTex, Irvine, CA).

Techniques: Knock-Out

Journal: American Journal of Physiology - Renal Physiology

Article Title: Furosemide reduces BK-αβ4-mediated K + secretion in mice on an alkaline high-K + diet

doi: 10.1152/ajprenal.00223.2018

Figure Lengend Snippet: Furosemide (Furo) effect on large conductance Ca2+-activated K+ (BK)-β4 expression in the kidney. A: BK-β4 expression in the kidney medulla of wild-type (WT) on high-K diet (HK) treated with control (Ctrl) or Furo water; n = 4/group. B: BK-β4 expression in the kidney cortex of WT on HK treated with Ctrl vs. Furo water. *P < 0.05 vs. HK + Ctrl analyzed with Student’s t-test; n = 7/group. C: BK-β4 expression in the kidney cortex of WT on HK treated with Ctrl vs. Furo acetazolamide (Actz) water; n = 4/group.

Article Snippet: Primary antibodies included anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories, Jerusalem, Israel), anti-NHE3 (mouse monoclonal, diluted 1:200; Invitrogen, Carlsbad, CA), and anti-V-ATPase B1 (rabbit polyclonal, diluted 1:200; GeneTex, Irvine, CA).

Techniques: Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Furosemide reduces BK-αβ4-mediated K + secretion in mice on an alkaline high-K + diet

doi: 10.1152/ajprenal.00223.2018

Figure Lengend Snippet: Furosemide concentrations in urine and plasma of WT and BK-β4-KO

Article Snippet: Primary antibodies included anti-BK-β4 (rabbit polyclonal, diluted 1:500; Alomone Laboratories, Jerusalem, Israel), anti-NHE3 (mouse monoclonal, diluted 1:200; Invitrogen, Carlsbad, CA), and anti-V-ATPase B1 (rabbit polyclonal, diluted 1:200; GeneTex, Irvine, CA).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mitochondrial accumulation of polyubiquitinated proteins and differential regulation of apoptosis by polyubiquitination sites Lys‐48 and ‐63

doi: 10.1111/j.1582-4934.2009.00775.x

Figure Lengend Snippet: (A) Activation of mitochondrial apoptosis by MG‐132. N27 cells were exposed to 2.5 μM MG‐132 for 5 min. and then chymotrypsin‐like proteasomal activity was assayed as described in the methods. The data represent mean ± S.E.M., n= 6, ***P < 0.001. (B) Cytochrome c release. N27 cells were treated with 2.5 μM MG‐132 for 45 or 90 min., and the level of cytosolic cytochrome c was examined by Western blot using cytochrome c antibody. The membranes were reprobed with β‐actin antibody. (C) Caspase‐9 activation. Caspase‐9 activity was assayed in the cells exposed to MG‐132 for 90 or 120 min. as described in the methods section. The activity was expressed as the percentage of vehicle‐treated cells. n= 6, ***P < 0.001 as compared to control group.

Article Snippet: The antibodies used included cytochrome c (BD Pharmingen, San Jose, CA, USA), β‐actin, (Sigma, St. Louis, MO, USA), COX IV (cytochrome c oxidase, subunit 4; Invitrogen, Carlsbad, CA, USA), ubiquitin (DAKO, Carpinteria, CA, USA) and voltage‐dependent anion channel (VDAC; Abcam, Cambridge, MA, USA).

Techniques: Activation Assay, Activity Assay, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mitochondrial accumulation of polyubiquitinated proteins and differential regulation of apoptosis by polyubiquitination sites Lys‐48 and ‐63

doi: 10.1111/j.1582-4934.2009.00775.x

Figure Lengend Snippet: Mitochondrial accumulation of ubiquitinated proteins. (A) Subcellular distribution of polyubiquitin proteins. N27 cells were treated with 2.5 μM MG‐132 for 40 min. The cytosolic, mitochondrial, nuclear and microsomal fractions were prepared as described in the methods. Equal amounts of proteins were used for Western blot analysis using antibodies against ubiquitin, and subcellular markers β‐actin (cytosol), COX IV (mitochondria), lamin B (Nuclei) and calreticulin (microsome). (B) Time‐dependent elevation of polyubiquitinated proteins in mitochondria. Cytosolic and mitochondrial fractions were prepared from N27 cells exposed to 2.5 μM MG‐132 for 20, 40 or 60 min., and processed for ubiquitin immunoblotting. (C) Verification of mitochondria ubiquitination by sucrose gradient. Crude mitochondria isolated from N27 cells exposed to MG‐132 (2.5 μM for 40 min.) were subjected to sucrose gradient separation as described in the methods section. All the fractions collected were resolved on SDS‐PAGE and blotted with ubiquitin, β‐actin and/or COX IV antibodies. (D) Dissociation of ubiquitin conjugates from mitochondrial outer membrane. Mitochondria isolated from vehicle (Con) or MG‐132‐treated cells (MG) were incubated with a high pH buffer as described in the methods section, and then mitochondria were spun down. Both the mitochondrial pellets (P) and the equal proportion of the supernatant (S) were separated on SDS‐PAGE and immunoblotted with antibodies for ubiquitin, VDAC and COX IV. (E) Ubiquitination of mitochondrial proteins in vitro. The reaction was carried out by incubating the mitochondrial suspension (4 mg/ml) with ubiquitination enzymes (9.6 μg for fraction A and B included in the kits), ubiquitin (8 μg), ubiquitin aldehyde and an energy source. Mitochondria were then recovered from the reaction mixture for Western blotting using ubiquitin or cytochrome c antibodies.

Article Snippet: The antibodies used included cytochrome c (BD Pharmingen, San Jose, CA, USA), β‐actin, (Sigma, St. Louis, MO, USA), COX IV (cytochrome c oxidase, subunit 4; Invitrogen, Carlsbad, CA, USA), ubiquitin (DAKO, Carpinteria, CA, USA) and voltage‐dependent anion channel (VDAC; Abcam, Cambridge, MA, USA).

Techniques: Western Blot, Isolation, SDS Page, Incubation, In Vitro

Journal: Journal of Cellular and Molecular Medicine

Article Title: Mitochondrial accumulation of polyubiquitinated proteins and differential regulation of apoptosis by polyubiquitination sites Lys‐48 and ‐63

doi: 10.1111/j.1582-4934.2009.00775.x

Figure Lengend Snippet: Effect of mutant ubiquitin on apoptosis. (A) Cytochrome c release. N27 cells stably expressing wt or mutant ubiquitins (K48R or K63R) were treated with 2.5 μM MG‐132 for 45 min., and the cytosolic fractions were subjected to Western blot analysis using cytochrome c. The membranes were reprobed with β‐actin and COX 4 antibodies. (B) Caspase‐9 activity, (C) Caspase‐3 activity and (D) DNA fragmentation. N27 cells stably expressing wt or mutant ubiquitins (K48R or K63R) were treated with MG‐132 for 120 min. Enzymatic activities of caspase‐9 and ‐3 and DNA fragmentation were determined as described previously. Data for caspase‐9 represent results of two separate experiments with a total of 11 individual samples, respectively; the data for caspase‐3 were derived from three experiments with a total of 16 individual samples, respectively. Two separate experiments with a total of six separate samples were analysed for DNA fragmentation. Values were expressed as the percentage of vehicle‐treated wt ubiquitin control group. **P < 0.01, ***P < 0.001 compared with individual control groups; ##P < 0.01, ###P < 0.001, comparison between indicated groups.

Article Snippet: The antibodies used included cytochrome c (BD Pharmingen, San Jose, CA, USA), β‐actin, (Sigma, St. Louis, MO, USA), COX IV (cytochrome c oxidase, subunit 4; Invitrogen, Carlsbad, CA, USA), ubiquitin (DAKO, Carpinteria, CA, USA) and voltage‐dependent anion channel (VDAC; Abcam, Cambridge, MA, USA).

Techniques: Mutagenesis, Stable Transfection, Expressing, Western Blot, Activity Assay, Derivative Assay

Journal: Skeletal Muscle

Article Title: Role of Parkin and endurance training on mitochondrial turnover in skeletal muscle

doi: 10.1186/s13395-018-0157-y

Figure Lengend Snippet: Effect of training and Parkin on whole muscle protein markers. a Representative Western blot of Parkin expression in untrained and trained skeletal muscle of control wild-type (WT) mice above. A graphical representation is shown below ( n = 5, * P < 0.05, vs UT). b Representative immunoblots of COXI, COXII, COXIV and TFAM expression in untrained and trained skeletal muscle of Parkin KO and WT animals. c A graphical representation of whole muscle COXI shown ( n = 4, * P < 0.05, main effect of training; # P < 0.05, vs trained WT). d Quantification of whole muscle COXII ( n = 6, * P < 0.05, main effect of training). e Whole muscle COXIV expression ( n = 4, * P < 0.05, main effect of training, # P < 0.05, main effect of genotype). f Quantification of whole muscle TFAM ( n = 4, * P < 0.05, main effect of training). Values are means ± SEM. † P < 0.05, interaction effect of training and genotype. α-tubulin was used for muscle loading control. WT, wild type; KO, Parkin knockout; UT, untrained; T, trained; COXI, cytochrome c oxidase subunit 1; COXII, cytochrome c oxidase subunit 2; COXIV, cytochrome c oxidase subunit 4; TFAM, mitochondrial transcription factor A; A.U., arbitrary units

Article Snippet: Blots were incubated overnight at 4 °C with primary antibodies against LC3/microtubule-associated protein light chain 3 (4108, Lot No. 3, Cell Signaling), p62/sequestosome 1 (P0067, Lot No. 015M4877V, Sigma), Parkin (4211, Lot No. 4, Cell Signaling), PGC-1α/peroxisome proliferator-activated receptor-γ coactivator-1α (ab3242, Lot No. 2691399, Millipore), PARIS/Parkin-interacting substrate (ab130867, Lot No. GR235090-1, abcam), phospho-acetyl-CoA carboxylase (07-303, Lot No. DAM1661071, Millipore), COXI/cytochrome c oxidase subunit 1 (ab14705, Lot No. GR233531-3, Abcam), COXII/cytochrome c oxidase subunit 2 (bs-2376R, Lot No. YEYL31W, Bioss), COXIV/cytochrome c oxidase subunit 4 (ab140643, Lot No. GR192963-3, Abcam), TFAM/mitochondrial transcription factor A (in house), VDAC/voltage-dependent anion channel (ab14734, Lot No. GR121056-7, Abcam), GAPDH (ab8245, Lot No. GR3185172-2, Abcam), and α-tubulin (CP06, Lot No. D00175772, Millipore).

Techniques: Western Blot, Expressing, Knock-Out

Journal: Skeletal Muscle

Article Title: Role of Parkin and endurance training on mitochondrial turnover in skeletal muscle

doi: 10.1186/s13395-018-0157-y

Figure Lengend Snippet: Mitochondrial adaptations following 6 weeks of voluntary wheel training. a Representative Western blots of COXI, COXII, COXIV and TFAM expression on isolated mitochondria from untrained and trained skeletal muscle of Parkin KO and WT animals. b Graphical representation of mitochondrial COXI expression ( n = 8). c Quantification of mitochondrial COXII content ( n = 4). d Mitochondrial yield of mixed hindlimb muscles of Parkin KO and WT animals ( n = 4). e Skeletal muscle cytochrome c oxidase (COX) activity following training in quadriceps muscle of Parkin KO and WT animals ( n = 5). f Mitochondrial state 4 and state 3 respiration rates in KO compared with WT animals ( n = 8). g Mitochondrial ROS emission expressed per natom of oxygen consumed in Parkin KO and WT mice ( n = 8; ¶ P < 0.05, vs state 4 trained WT). Values are means ± SEM. * P < 0.05, main effect of training; # P < 0.05, main effect of genotype; † P < 0.05, interaction effect of genotype and training. Voltage-dependent anion channel (VDAC) was used as a mitochondrial loading control. WT, wild type; KO, Parkin knockout; UT, untrained; T, trained; COX1, cytochrome c oxidase subunit 1; COXII, cytochrome c oxidase subunit 2; COX4, cytochrome c oxidase subunit 4; TFAM, mitochondrial transcription factor A; ROS, reactive oxygen species; A.U., arbitrary units

Article Snippet: Blots were incubated overnight at 4 °C with primary antibodies against LC3/microtubule-associated protein light chain 3 (4108, Lot No. 3, Cell Signaling), p62/sequestosome 1 (P0067, Lot No. 015M4877V, Sigma), Parkin (4211, Lot No. 4, Cell Signaling), PGC-1α/peroxisome proliferator-activated receptor-γ coactivator-1α (ab3242, Lot No. 2691399, Millipore), PARIS/Parkin-interacting substrate (ab130867, Lot No. GR235090-1, abcam), phospho-acetyl-CoA carboxylase (07-303, Lot No. DAM1661071, Millipore), COXI/cytochrome c oxidase subunit 1 (ab14705, Lot No. GR233531-3, Abcam), COXII/cytochrome c oxidase subunit 2 (bs-2376R, Lot No. YEYL31W, Bioss), COXIV/cytochrome c oxidase subunit 4 (ab140643, Lot No. GR192963-3, Abcam), TFAM/mitochondrial transcription factor A (in house), VDAC/voltage-dependent anion channel (ab14734, Lot No. GR121056-7, Abcam), GAPDH (ab8245, Lot No. GR3185172-2, Abcam), and α-tubulin (CP06, Lot No. D00175772, Millipore).

Techniques: Western Blot, Expressing, Isolation, Activity Assay, Knock-Out