Journal: Amyotrophic Lateral Sclerosis & Frontotemporal Degeneration

Article Title: Immunohistochemical detection of C9orf72 protein in frontotemporal lobar degeneration and motor neurone disease: patterns of immunostaining and an evaluation of commercial antibodies

doi: 10.1080/21678421.2017.1359304

Figure Lengend Snippet: Antibodies employed in the study.

Article Snippet: The other two Proteintech polyclonal antibodies (18450 and 23058) barely immunostained such cells, even at high antibody concentrations (not shown).

Techniques:

Journal: Amyotrophic Lateral Sclerosis & Frontotemporal Degeneration

Article Title: Immunohistochemical detection of C9orf72 protein in frontotemporal lobar degeneration and motor neurone disease: patterns of immunostaining and an evaluation of commercial antibodies

doi: 10.1080/21678421.2017.1359304

Figure Lengend Snippet: Immunostaining of Purkinje cells of the cerebellum for C9orf72 protein using C9-L antibody (a), C9-S antibody (b), Proteintech monoclonal antibody (c), Proteintech polyclonal antibody, 20422 (d), GeneTex antibody (e), Proteintech polyclonal antibodies, 18450 (f) and 23058 (g), Santa Cruz antibody (h) and Abgent antibody (i). Immunoperoxidase–haematoxylin, ×400 microscope magnification.

Article Snippet: The other two Proteintech polyclonal antibodies (18450 and 23058) barely immunostained such cells, even at high antibody concentrations (not shown).

Techniques: Immunostaining, Microscopy

Journal: Amyotrophic Lateral Sclerosis & Frontotemporal Degeneration

Article Title: Immunohistochemical detection of C9orf72 protein in frontotemporal lobar degeneration and motor neurone disease: patterns of immunostaining and an evaluation of commercial antibodies

doi: 10.1080/21678421.2017.1359304

Figure Lengend Snippet: Immunostaining of anterior horn cells of the spinal cord for C9orf72 protein using C9-L antibody (a), Proteintech monoclonal antibody (b), Proteintech polyclonal antibody, 20422 (c), GeneTex antibody (d), Santa Cruz antibody (e) and Abgent antibody (f). Immunoperoxidase–haematoxylin, ×400 microscope magnification.

Article Snippet: The other two Proteintech polyclonal antibodies (18450 and 23058) barely immunostained such cells, even at high antibody concentrations (not shown).

Techniques: Immunostaining, Microscopy

Journal: EMBO Reports

Article Title: MITRAC15/COA1 promotes mitochondrial translation in a ND2 ribosome–nascent chain complex

doi: 10.15252/embr.201948833

Figure Lengend Snippet: Solubilized mitochondria from wild‐type HEK293T cells were subjected to immunoisolation with MITRAC12 antiserum. Eluates were analyzed by SDS–PAGE and Western blotting. Total 1.5%, eluate 100%. Wild‐type mitochondria were solubilized and subjected to 2D‐BN/SDS–PAGE and Western blotting with indicated antibodies. Solubilized mitochondria from MITRAC12 FLAG ‐ or ACAD9 FLAG ‐expressing cells were subjected to anti‐FLAG immunoisolation and native elution. Eluates were analyzed by 2D‐BN/SDS–PAGE and Western blot analysis. Total 0.8% (MITRAC12 FLAG isolation), 0.35% (ACAD9 FLAG isolation), eluate 100%. Solubilized mitochondria from MITRAC15 FLAG ‐expressing cells were first subjected to native anti‐FLAG immunoisolation (total 0.1%, eluate 10%, lane 4). Purified complexes were applied to anti‐MITRAC12 immunoisolation (eluate 100%, lane 5). All samples were analyzed by SDS–PAGE and immunoblotting.

Article Snippet: Primary commercial antibodies used were as follows: ACAD9 (15770‐1‐AP, Proteintech, rabbit), ND2 (19704‐1‐AP, Proteintech, rabbit), mS40 (16139‐1‐AP, Proteintech, rabbit), NDUFB10 (ab196019, Abcam, rabbit), ND5 (55410‐1‐AP, Proteintech, rabbit), NDUFS1 (12444‐1‐AP, Proteintech, rabbit), NDUFAF4 (26003‐1‐AP, Proteintech, rabbit), NDUFA5 (16640‐1‐AP, Proteintech, rabbit), and NDUFB10 (ab192245, Abcam, rabbit).

Techniques: SDS Page, Western Blot, Expressing, Isolation, Purification

Journal: EMBO Reports

Article Title: MITRAC15/COA1 promotes mitochondrial translation in a ND2 ribosome–nascent chain complex

doi: 10.15252/embr.201948833

Figure Lengend Snippet: Mitochondrial translational products were labeled with [ 35 S]methionine, and anti‐FLAG immunoisolation was performed as described in Fig D. Eluates were analyzed by SDS–PAGE and digital autoradiography. Total 1.5%, eluate 100 %. Solubilized mitochondria from MITRAC15 FLAG ‐ or TIM21 FLAG ‐expressing cells were subjected to anti‐FLAG immunoisolation and analyzed by 2D‐BN/SDS–PAGE and Western blotting. MITRAC15 FLAG isolation: total 0.1%, eluate 100%. TIM21 FLAG isolation: total 0.2%, eluate 66% (TIM21 FLAG decoration), 33% (MITRAC15, ACAD9, MITRAC12, COX1 decoration). Solubilized mitochondria from MITRAC15 FLAG ‐ or C12ORF62 FLAG ‐ expressing cells were subjected to anti‐FLAG immunoisolation and analyzed by SDS–PAGE and immunoblotting. Total 0.4%, eluate 100% (MITRAC15 FLAG isolation), 70% (C12ORF62 FLAG isolation). Solubilized mitochondria from ACAD9 FLAG ‐ or MITRAC15 FLAG ‐ expressing cells were subjected to anti‐FLAG immunoisolation and analyzed by SDS–PAGE and immunoblotting. Total 0.5% (ACAD9 FLAG isolation), 0.4% (MITRAC15 FLAG isolation), eluate 100%. Asterisk (*) indicates TIM21 signal. ACAD9/ACAD9 FLAG could not be separated with the used gel system, both proteins are detected with ACAD9 antiserum.

Article Snippet: Primary commercial antibodies used were as follows: ACAD9 (15770‐1‐AP, Proteintech, rabbit), ND2 (19704‐1‐AP, Proteintech, rabbit), mS40 (16139‐1‐AP, Proteintech, rabbit), NDUFB10 (ab196019, Abcam, rabbit), ND5 (55410‐1‐AP, Proteintech, rabbit), NDUFS1 (12444‐1‐AP, Proteintech, rabbit), NDUFAF4 (26003‐1‐AP, Proteintech, rabbit), NDUFA5 (16640‐1‐AP, Proteintech, rabbit), and NDUFB10 (ab192245, Abcam, rabbit).

Techniques: Labeling, SDS Page, Autoradiography, Expressing, Western Blot, Isolation

Journal: EMBO Reports

Article Title: MITRAC15/COA1 promotes mitochondrial translation in a ND2 ribosome–nascent chain complex

doi: 10.15252/embr.201948833

Figure Lengend Snippet: Mitochondrial translation products were radiolabeled with [ 35 S]methionine after siRNA‐mediated depletion of ACAD9 in wild‐type and MITRAC15 −/− cells. Cell lysates were analyzed by SDS–PAGE and digital autoradiography. For quantifications, the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal (error bars indicate SEM from three biological replicates; * P < 0.05; ** P < 0.01; *** P < 0.001; unpaired t ‐test). FLAG immunoprecipitation was performed in mitochondrial extracts from MITRAC15 FLAG ‐expressing cells after siRNA‐mediated downregulation of ACAD9 and pulse radiolabeling. SDS–PAGE followed by autoradiography was used to analyze eluates. Relative signal intensity of ND2, ND3, and ND4L was normalized to ATP6 (total) or isolation efficiency (eluate). Total 2%, eluate 100%. Error bars indicate SEM from three biological replicates; ** P < 0.01; unpaired t ‐test. After C12orf62 downregulation, [ 35 S]methionine labeling was performed. During labeling, mitochondrial translation was inhibited with puromycin (pur) (2 μg/ml). MITRAC15 FLAG ‐interacting nascent chains were isolated by FLAG immunoprecipitation from cell lysates. Eluates were subjected to SDS–PAGE and analyzed by digital autoradiography. Total 2%, MITRAC15 FLAG eluate 100% (in case of C12ORF62 FLAG , 25% of eluate was loaded as a standard). Red asterisks, accumulating nascent chains of ND2. The accumulating nascent chains of ND2 of lane 12 are presented in a magnification. F1‐F3 mark nascent chains of COX1 . HEK293T cells were pulse‐labeled with [ 35 S]methionine in the presence of puromycin (2 μg/ml). During puromycin treatment, cells were incubated for 5 and 10 min in medium lacking [ 35 S]methionine. Anti‐FLAG immunoisolations were performed as described and eluates analyzed as described in (C). Total 1%, eluate 100%. The signal of ND2 was graphed after normalization against the COX2 signal, and the signal of mature ND2 in the 5‐min chase sample was set as 100% (error bars indicate SEM from three biological replicates; * P < 0.05; paired t ‐test). Red asterisks, accumulating nascent chains of ND2.

Article Snippet: Primary commercial antibodies used were as follows: ACAD9 (15770‐1‐AP, Proteintech, rabbit), ND2 (19704‐1‐AP, Proteintech, rabbit), mS40 (16139‐1‐AP, Proteintech, rabbit), NDUFB10 (ab196019, Abcam, rabbit), ND5 (55410‐1‐AP, Proteintech, rabbit), NDUFS1 (12444‐1‐AP, Proteintech, rabbit), NDUFAF4 (26003‐1‐AP, Proteintech, rabbit), NDUFA5 (16640‐1‐AP, Proteintech, rabbit), and NDUFB10 (ab192245, Abcam, rabbit).

Techniques: SDS Page, Autoradiography, Immunoprecipitation, Expressing, Radioactivity, Isolation, Labeling, Incubation

Journal: EMBO Reports

Article Title: MITRAC15/COA1 promotes mitochondrial translation in a ND2 ribosome–nascent chain complex

doi: 10.15252/embr.201948833

Figure Lengend Snippet: A, B Radiolabeling with [ 35 S]methionine, anti‐FLAG immunoisolation, and sample analyses were performed as described in Fig C. (B) Depletion of ACAD9 by siRNA was performed as in Fig A. Total 1%, eluate 100%. Red asterisks, accumulating nascent chains of ND2. C Identified crosslink between murine ACAD9 and ND2 (spectrum in Fig ). ND2: red—crosslinked peptide, green—C‐terminal helix, yellow—crosslinked Lys 319 . ACAD9: orange—crosslinked Lys 504 . Sequences of identified crosslinked peptides from mouse and the homologous sequences from the human proteins are presented (yellow: crosslinked lysine).

Article Snippet: Primary commercial antibodies used were as follows: ACAD9 (15770‐1‐AP, Proteintech, rabbit), ND2 (19704‐1‐AP, Proteintech, rabbit), mS40 (16139‐1‐AP, Proteintech, rabbit), NDUFB10 (ab196019, Abcam, rabbit), ND5 (55410‐1‐AP, Proteintech, rabbit), NDUFS1 (12444‐1‐AP, Proteintech, rabbit), NDUFAF4 (26003‐1‐AP, Proteintech, rabbit), NDUFA5 (16640‐1‐AP, Proteintech, rabbit), and NDUFB10 (ab192245, Abcam, rabbit).

Techniques: Radioactivity

Journal: EMBO Reports

Article Title: MITRAC15/COA1 promotes mitochondrial translation in a ND2 ribosome–nascent chain complex

doi: 10.15252/embr.201948833

Figure Lengend Snippet: A knockout cell line of MITRAC15 (MITRAC15 −/− ) was created in HEK293T cells by using CRISPR/Cas9 technology. Alignment of respective mutated region compared to wild type shows premature STOP codons (*) in MITRAC15 −/− . Mitochondria from wild‐type and MITRAC15 −/− cells were isolated and subjected to steady‐state protein analyses by Western blotting. Mitochondria from wild‐type and MITRAC15 −/− cells were isolated and analyzed by BN‐PAGE. Triton X‐100 (left site) or digitonin (right site) was used for solubilization. The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels (error bars indicate SEM from three biological replicates). Complex I and citrate synthase activity were measured in WT and MITRAC15 −/− cells (error bars indicate SEM from three biological replicates; * P < 0.05; unpaired t ‐test). Pulse‐chase [ 35 S]methionine radiolabeling of mitochondrial translation products in wild‐type and MITRAC15 −/− cells. Samples were analyzed by SDS–PAGE and digital autoradiography. Signal intensity of ND2 was normalized to the ATP6 signal, and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal (error bars indicate SEM from three biological replicates; * P < 0.05; unpaired t ‐test). Pulse radiolabeling after re‐expressing MITRAC15 FLAG in MITRAC15 −/− cells. Samples were analyzed by SDS–PAGE and digital autoradiography. Relative pulse signal intensity of ND2 was normalized to ATP6 (error bars indicate SEM from three biological replicates; * P < 0.05; ** P < 0.01; unpaired t ‐test).

Article Snippet: Primary commercial antibodies used were as follows: ACAD9 (15770‐1‐AP, Proteintech, rabbit), ND2 (19704‐1‐AP, Proteintech, rabbit), mS40 (16139‐1‐AP, Proteintech, rabbit), NDUFB10 (ab196019, Abcam, rabbit), ND5 (55410‐1‐AP, Proteintech, rabbit), NDUFS1 (12444‐1‐AP, Proteintech, rabbit), NDUFAF4 (26003‐1‐AP, Proteintech, rabbit), NDUFA5 (16640‐1‐AP, Proteintech, rabbit), and NDUFB10 (ab192245, Abcam, rabbit).

Techniques: Knock-Out, CRISPR, Isolation, Western Blot, Activity Assay, Pulse Chase, Radioactivity, SDS Page, Autoradiography, Expressing

Journal: Cell Death & Disease

Article Title: Avenanthramide A triggers potent ROS-mediated anti-tumor effects in colorectal cancer by directly targeting DDX3

doi: 10.1038/s41419-019-1825-5

Figure Lengend Snippet: a Schematic representation of the truncation mutants of human DDX3. NTD, N-terminal regulatory domain; Helicase, Helicase domain; CTD, C-terminal regulatory domain. b Three truncation mutants of DDX3 were purified and determined by SDS-PAGE. c SPR assay was used to evaluate the binding affinities of three truncation mutants of DDX3 to AVN A. d Mutations K255A, R287A, S290A, and R294A in the ATP-binding pocket of DDX3 were performed by site-directed mutagenesis. DLD1 cells were transfected with wild-type DDX3 or the four site mutants, and then treated with 50 μg/ml AVNs or 30 μM AVN A, after 24 h cell viabilities were detected by MTT assay ( n = 3, mean ± SD). e The expression of NDUFS2 and UQCRC1 in wild-type or the 4 DDX3 site mutants were determined by western blot. f The levels of ROS in wild-type or the four site mutants of DDX3 treated with 30 μM AVN A were measured by flow cytometry. * P < 0.05, ** P < 0.01, *** P < 0.001. P -values were calculated by a Student’s t -test. All experiments have been replicated three independent times

Article Snippet: The antibodies against NDUFS2, UQCRC1, ND2, ND5, CYTB, COX2, ATP6, and ATP5A1 were purchased from Proteintech (Wuhan, China).

Techniques: Purification, SDS Page, SPR Assay, Binding Assay, Mutagenesis, Transfection, MTT Assay, Expressing, Western Blot, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: ING2 positively regulates mitochondrial respiration in tubular epithelial cells. HK2 cells were transfected with ING2-shRNA or ING2-overexpression plasmid. (A,B) The transfection efficiencies were evaluated by qPCR and Western blotting, respectively. (C–F) Following transfection, mitochondrial OXPHOS of HK2 cells were detected using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A&R, antimycin and rotenone. (G,H) mtDNA-encoded components for complex I, III, IV, and V and 16S rRNA were determined by qPCR. (I) mtDNA-encoded components for complex I, III, IV, and V were determined by Western blotting. (J) The mitochondria were stained with MitoTracker (red) and ING2 (green) followed by DAPI (blue) re-dyeing. (K) The mitochondria DNA (mtDNA) copy number was determined by qPCR with G6PC serving as internal reference. Data were from three individual experiments and presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns, not significant.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Transfection, shRNA, Over Expression, Plasmid Preparation, Western Blot, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: MRPL12 mediated the effects of ING2 on mitochondrial OXPHOS in tubular epithelial cells. (A,B) HK2 cells were transfected with ING2-shRNA or ING2-overexpression plasmid. The mRNA and protein levels of TFAM and MRPL12 were evaluated by qPCR and Western blotting. (C,D) HK2 cells were divided into control group, ING2 overexpression group, MRPL12 knockout group, and MRPL12 knockout plus ING2 overexpression group. Mitochondrial OXPHOS was detected using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A&R, antimycin and rotenone. (E) Western blotting to validate the altered expression of mtDNA-encoded components of mitochondria complex. Data were from three individual experiments and presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Transfection, shRNA, Over Expression, Plasmid Preparation, Western Blot, Control, Knock-Out, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: ING2 inhibited the ubiquitination of MRPL12. (A) HK2 cells were transfected with control or ING2-shRNA, followed by MG132 treatment for 6 h. Protein levels of ING2 and MRPL12 were evaluated by Western blotting. (B) A ubiquitin modified site was found at 58 lysine residue (red, upper panel), predicted using predictor of protein ubiquitination sites, UbPred. (C,D) The ubiquitination of MRPL12 was determined by proximity ligation assay (PLA) and immunoprecipitation, respectively, after transfected ING2-overexpression plasmid. (E) By using ASEB, a web tool for predicting protein acetylation site, a acetylation site was detected at the 185 lysine residue (green, lower panel). (F) The acetylation of MRPL12 was determined by immunoprecipitation after transfected ING2-overexpression plasmid. IP, immunoprecipitation; WB, Western blotting. Data were from three individual experiments.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Ubiquitin Proteomics, Transfection, Control, shRNA, Western Blot, Modification, Residue, Proximity Ligation Assay, Immunoprecipitation, Over Expression, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: ING2 ameliorated the ischemia induced mitochondrial OXPHOS defects and tubular cell apoptosis. (A) Immunostaining of kidney tissue section from healthy individual and AKI patients with antibodies against ING2. At least two patients for each. (B) Immunostaining of kidney tissue section from healthy and AKI mice with antibodies against ING2 and MRPL12. HK2 cells were subjected to serum deprivation for 48 h, and protein contents of both ING2, MRPL12, and mtDNA-encoded components of mitochondria complex (C) were figured out by Western blotting. The apoptosis of HK2 cells was evaluated by flow cytometry (D) . HK2 cells were divided into control group, ING2 overexpression group, serum deprivation group, and serum deprivation plus ING2 overexpression group. MRPL12 and mtDNA-encoded components of mitochondria complex were detected through Western blotting (E) . Mitochondrial OXPHOS was detected using Seahorse (F,G) . The apoptosis of HK2 cells was evaluated by flow cytometry (H) , and the ubiquitination of MRPL12 was determined by proximity ligation assay (PLA) (I) . OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A&R, antimycin and rotenone. Data were from three individual experiments and presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Immunostaining, Western Blot, Flow Cytometry, Control, Over Expression, Ubiquitin Proteomics, Proximity Ligation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: ING2 overexpression effectively ameliorated ischemic kidney injury. The mice were injected with rAAV-con or rAAV-ING2 followed by the induction of IRI with sham operation group as control. (A) Immunofluorescent staining of kidneys after the intra-renal injection of rAAV vectors with ING2 (red) and DAPI (blue). (B) Immunostaining of renal tissue sections with antibodies against ING2, MRPL12, ND2, and COX II. (C) Western blot analysis of renal tissue ING2, MRPL12, ND2, and COX II. (D) Levels of creatinine in the serum or urine of IRI and sham operation mice. Data were from two individual experiments and presented as mean ± SEM. rAAV-con + IRI, n = 6; rAAV-ING2 + IRI, n = 6; rAAV-con + sham, n = 4; rAAV-ING2 + sham, n = 4. ** p < 0.01. ns, not significant.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Over Expression, Injection, Control, Staining, Immunostaining, Western Blot

Journal: Redox biology

Article Title: Decreased expression of mitochondrial aminoacyl-tRNA synthetases causes downregulation of OXPHOS subunits in type 2 diabetic muscle.

doi: 10.1016/j.redox.2023.102630

Figure Lengend Snippet: Fig. 2. Skeletal muscle gene expression of mitochondrial tRNA synthetases in early-onset type 2 diabetic subjects (YT2) and late-onset type 2 diabetic subjects (OT2) and their respective matched control groups (YC and OC). Real-time PCR was performed in skeletal muscle biopsies. YT2 are early-onset type 2 diabetic subjects; OT2 are late-onset type 2 diabetic subjects, and their respective controls are YC and OC. (A) CARS2, (B) DARS2, (C) HARS2, (D) IARS2, (E) LARS2, (F) MARS2, (G) NARS2, (H) PARS2, (I) SARS2. Data are presented in boxplots; on each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 74th percentiles, respectively. Unpaired non-parametric distribution and Mann-Whitney test was used *p < 0.05. YC (n = 12), YT2 (n = 21), OC (n = 17), OT2 (n = 24).

Article Snippet: The following antibodies were used: Oxphos (abcam ab110413), ND2 (Proteintech 16879184), ATP6 (Millipore MABS1995), VDAC/Porin (Santa cruz 73614), Vinculin (Santa cruz 73614), LARS2 (Proteintech 17097-1- AP), TARS2 (Proteintech 15067-1-AP), NOS2 (Santa cruz sc-7271), NOS3 (BD Transduction 610296).

Techniques: Gene Expression, Control, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Redox biology

Article Title: Decreased expression of mitochondrial aminoacyl-tRNA synthetases causes downregulation of OXPHOS subunits in type 2 diabetic muscle.

doi: 10.1016/j.redox.2023.102630

Figure Lengend Snippet: Fig. 3. Skeletal muscle gene expression of mitochondrial tRNA synthetases in type 2 diabetic subjects (T2D) and their respective matched control groups. Real-time PCR was performed in skeletal muscle biopsies. T2D are type 2 diabetic subjects and their respective matched control groups. (A) CARS2, (B) DARS2, (C) HARS2, (D) IARS2, (E) LARS2, (F) MARS2, (G) NARS2, (H) PARS2, (I) SARS2. Data are presented in boxplots; on each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 74th percentiles, respectively. Statistical analyses comparing type 2 diabetic subjects vs respective controls were performed by unpaired t-test *p < 0.05. C (n = 29), T2D (n = 45).

Article Snippet: The following antibodies were used: Oxphos (abcam ab110413), ND2 (Proteintech 16879184), ATP6 (Millipore MABS1995), VDAC/Porin (Santa cruz 73614), Vinculin (Santa cruz 73614), LARS2 (Proteintech 17097-1- AP), TARS2 (Proteintech 15067-1-AP), NOS2 (Santa cruz sc-7271), NOS3 (BD Transduction 610296).

Techniques: Gene Expression, Control, Real-time Polymerase Chain Reaction

Journal: Redox biology

Article Title: Decreased expression of mitochondrial aminoacyl-tRNA synthetases causes downregulation of OXPHOS subunits in type 2 diabetic muscle.

doi: 10.1016/j.redox.2023.102630

Figure Lengend Snippet: Fig. 4. Skeletal muscle gene and protein expression of mitochondrial tRNA synthetases in obese and diabetic mice and their respective matched control group. Real-time PCR and Western blot were performed in skeletal muscle biopsies. Ob/ob are obese mice, db/db are diabetic mice, and their respective controls are C57BL/ 6 and db/+. (A) Skeletal muscle gene expression of mt-aaRSs in obese mice. (B) Skeletal muscle gene expression of mt-aaRSs in diabetic mice. (C) Skeletal muscle protein expression of LARS2 and TARS2 in obese mice. (D) Skeletal muscle protein expression of LARS2 and TARS2 in diabetic mice. Data are mean ± SEM. Sta tistical analyses comparing C57BL6 vs ob/ob or db/+ vs db/db mice were performed by unpaired t-test *p < 0.05. n = 4–6/group.

Article Snippet: The following antibodies were used: Oxphos (abcam ab110413), ND2 (Proteintech 16879184), ATP6 (Millipore MABS1995), VDAC/Porin (Santa cruz 73614), Vinculin (Santa cruz 73614), LARS2 (Proteintech 17097-1- AP), TARS2 (Proteintech 15067-1-AP), NOS2 (Santa cruz sc-7271), NOS3 (BD Transduction 610296).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Western Blot, Gene Expression