Journal: Virology

Article Title: Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

doi: 10.1016/j.virol.2018.08.008

Figure Lengend Snippet: (A) stain-free blot representing the spectrum of proteins obtained for HCMV and EV fractions after separation of AD169 HCMV pre-paration using a iodixanol step-gradient centrifugation. (B) Detection of EV-associated proteins CD63 (~60 kDa), Rab27A (~30 kDa), and calnexin (~25 kDa) proteins in both fractions. (C) Detection of HCMV capsid protein MCP (~150 kDa) and HCMV tegument protein, pp150 (~160 kDa) in HCMV and EV fractions after separation of AD169 viral preparation using a iodixanol step-gradient centrifugation.

Article Snippet: 10 μg of proteins were loaded on a 4–20% precast polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and separated by SDS-PAGE, then transferred to PVDF membranes and probed with anti-CD63 (1 μg/ml, Thermo Fisher Scientific, Waltham, MA), anti-Calnexin (1 μg/ml, Thermo Fisher Scientific, Waltham, MA), anti-Rab27A (1 μg/ml, Thermo Fisher Scientific, Waltham, MA), anti-HCMV capsid MCP (2 μg/ml), and anti-HCMV tegument pp150 (2 μg/ml) monoclonal primary anti-mouse monoclonal antibodies (clones 28–4 and 36–14) and then goat peroxidase-conjugated anti-mouse IgG secondary anti-body (Bio-Rad Laboratories, Hercules, CA).

Techniques: Staining, Gradient Centrifugation

Journal: Cells

Article Title: PKH high /CD133+/CD24− Renal Stem-Like Cells Isolated from Human Nephrospheres Exhibit In Vitro Multipotency

doi: 10.3390/cells9081805

Figure Lengend Snippet: Morphogenesis 3D assay. ( a ) Contrast-phase images of RSC (left), CD31−/PKH low/neg (middle) and CD31+/PKH low/neg (right) at 6 h of culture. Scale bars, 100 µm; ( b ) contrast-phase images of structures obtained in Matrigel at 6–7 days of culture from the 3 different samples #1, #2, #3 of RSC (top), CD31−/PKH low/neg (middle), CD31+/PKH low/neg (bottom). Scale bars, 100 µm; ( c ) percentage of structures with sprouts in the three different samples of CD31+/PKH low/neg at different time points; ( d ) 3D IF staining of the structures obtained from CD31+/PKH low/neg cells with the antibodies against the indicated markers. Two different fields for each staining are shown. CK: cytokeratin; ECAD: E-cadherin; vWf: von Willebrand Factor; Blue—DAPI. Original magnification 400×. Scale bars, 50 µm. Arrows: CD31+ cells in the sprout-like extensions.

Article Snippet: IF staining of histologic sections was performed as described [ ] , using mouse monoclonal anti-aquaporin (AQP1, clone B-11, Santa Cruz, Dallas, TX, USA, 1:50), rabbit polyclonal anti-CD13 (Santa Cruz, Dallas, TX, USA, 1:200), mouse monoclonal anti-N-cadherin (NCAD, clone 32, Becton Dickinson, San Jose, CA, USA, 1:50) proximal tubular markers; rabbit monoclonal anti-cytokeratin 7 (CK7, clone EPR1619Y, Abcam; 1:200), mouse monoclonal anti-calbindin-D28k (CALB, clone CB-955, Sigma-Aldrich, St.Louis, MO, USA, 1:200), mouse monoclonal anti- E-cadherin (ECAD, clone 36, Becton Dickinson, San Jose, CA, USA, 1:50) distal tubular markers; mouse monoclonal anti-cytokeratin 8.18 (CK 8.18), epithelial marker (clone 5D3, 1:50); von Willebrand factor (vWf), endothelial marker (1:2000) primary antibodies and Alexa Fluor 680 conjugated goat anti-mouse and Alexa Fluor 594 conjugated goat anti-rabbit IgG secondary antibodies (1:100).

Techniques: Staining

Journal: Oncogene

Article Title: ZEB1-repressed microRNAs inhibit autocrine signaling that promotes vascular mimicry of breast cancer cells

doi: 10.1038/onc.2017.356

Figure Lengend Snippet: Increased expression of FN1 and SERPINE2 is regulated by serum withdrawal and is essential for VM. ( a ) Quantitative reverse transcription–PCR (qRT–PCR) in breast cancer cell lines cultured with 10 or 0% serum for 48 h. Error bars represent s.d. of three independent experiments. T -tests were performed for each line comparing 0vs10% conditions, ** P <0.01, *** P <0.001. ( b , d ) Representative images of VM in control and FN1 ( b ) or SERPINE2 ( d ) knockdown cells. Scale bars=1000 μm. ( c , e ) Quantification of total network length for VM assays as shown in b and d . Error bars represent s.d. for at five and four independent experiments, respectively. T -tests were performed to compare each to siNT control, ** P <0.01. ( f ) Representative images of VM in control and FN1 or SERPINE2 knockdown in BT-549 cells. Scale bars=1000 μm. ( g ) Quantification of total network length. Error bars represent s.d. for three independent experiments. T -tests were performed to compare each to siNT control, * P <0.05. ( h ) Representative phase images of cell morphology in siNT or siSERPINE2 cells. Scale bars=100 μm. ( i ) qRT–PCR for FN1 and CDH1 in cells transfected with siNT or siSERPINE2. Error bars represent s.d. of three independent experiments. T -test was performed, *** P <0.001. ( j ) qRT–PCR for FN1 and SERPINE2 in cells transfected with NEG or clustered miRNA mimics for 48 h, then cultured with 10 or 0.5% serum for 48 h. Error bars represent s.d. of three independent experiments. T -tests were performed comparing 0.5vs10% in the control or comparing cells transfected with miRNAs vs NEG in matching serum conditions, *** P <0.001. ( k ) Western blot for FN1 for cells as in h . ACTB is loading control. ( l , m ) Quantification of total network length of VM assays from VM-incompetent cells plated in 0% serum ( l ) or VM-competent cells plated in 5% serum ( m ) in the presence or absence of recombinant FN1 and SERPINE2 as shown. Error bars represent s.d. of two independent experiments.

Article Snippet: Primary antibodies used were ZEB1 (H-102, Santa Cruz Biotechnology, Santa Cruz, CA, USA, used at a 1:200 dilution), SEC23A (NBP1-32773, Novus Biologicals, Littleton, CO, USA, used at a 1:5000 dilution), CDH1 (36/E-cadherin, BD Biosciences, used at a 1:5000 dilution), FN1 (C-20, Santa Cruz Biotechnology, used at a 1:100 or 1:200 dilution), SERPINE2 (8C4.1, Millipore, used at 1:1000), LRP1 (EPR3724, Abcam, Cambridge, UK, used at 1:5000), GAPDH (6C5, Thermo Fisher, used at 1:10 000) and ACTB (AC-15, Sigma-Aldrich, St. Louis, MO, USA, used at a 1:10 000 dilution).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Transfection, Western Blot, Recombinant

Journal: PLoS ONE

Article Title: Epiplakin Deficiency Aggravates Murine Caerulein-Induced Acute Pancreatitis and Favors the Formation of Acinar Keratin Granules

doi: 10.1371/journal.pone.0108323

Figure Lengend Snippet: (A) Immunofluorescence microscopy for K8 in pancreata from wild-type and EPPK −/− mice reveal no differences in keratin network structure of acinar cells. (B–E) Immunofluorescence microscopy analysis using antibodies to desmoplakin (B, C) e-cadherin (D, E) and occludin (F, G) shows no differences in intensity and localization of these junctional proteins between wild-type and EPPK −/− mice in unstressed tissue (B, D, F) and during pancreatitis (C, E, G). Note the disassembly of desmoplakin- and occludin-positive structures during pancreatitis in both wild-type and EPPK −/− mice (B–E). Scale bars, 20 µm.

Article Snippet: The following primary antibodies were used for immunoblotting (IB), immunofluorescence microscopy (IFM) and immunohistochemistry (IHC): affinity-purified rabbit antibodies to epiplakin (IB 1∶10.000, IFM 1∶1.000, IHC 1∶1000) ; mouse monoclonal antibody (mAb) to K18 (Ks18.04, Progen; IB 1∶500); rabbit mAb to K7 (R17-S, DB Biotech, Kosice, Slovakia; IFM 1∶100); rat mAb to K8 (Troma I, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA ; IB 1∶500, IFM 1∶50, IHC 1∶50); mAb to desmoplakin (DP I/II 236.23.1, Progen; IFM undiluted); mAb to occludin (OC-3F10, Life Technologies, Carlsbad, CA; IFM 1∶200); mAb to e-cadherin (36/E-Cadherin, BD Biosciences, Franklin Lakes, NJ; IFM 1∶35); and rat mAb to K19 (Troma III, Developmental Studies Hybridoma Bank, University of Iowa ; IB 1∶500).

Techniques: Immunofluorescence, Microscopy

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 1. Vitamin D3 increases legumain expression, activity, and secretion in pre-osteoblastic cells. (A) The nucleotide sequence of the LGMN gene promoter region with annotations of potential vitamin D-responsive elements (VDRE; red) relative to the transcription start site (TSS). (B–F) Human BMSC- TERT cells (20,000 cells/cm2) were incubated with 1,25(OH)2D3 (B–F; 10, 50 or 100 nM), 25(OH)D3 (C–F; 100, 250, 500 or 1000 nM) or an equal volume of ethanol (control, 0 nM) in osteoblast induction medium for seven days before harvesting. (B) Legumain mRNA expression relative to housekeeping control (GAPDH) (2−∆∆CT; n = 3). (C) One representative immunoblot of legumain (proform 56 kDa, mature form 36 kDa) and GAPDH (housekeeping) in cell lysates (n = 3). (D) Quantification of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in C (n = 3). (E) Legumain activity (dF/s) in cell lysates adjusted for the total protein concentration (µg/mL) (n = 6–9). (F) Secreted legumain (pg/mL) in conditioned media measured by ELISA and adjusted for the total protein concentration in the corresponding cell lysates (n = 3–5). (B,D–F) Data represent mean ± SEM. (B,D) Kruskal–Wallis test. (E,F) One-way ANOVA. * p < 0.05 vs. 0 nM 1,25(OH)2D3 or 25(OH)D3. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Sequencing, Incubation, Control, Western Blot, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 2. Treatment with 25(OH)D3 increases legumain levels and activity in wild-type mice. Wild-type mice (Lgmn+/+) were treated with 50 µg/kg 25(OH)D3 (n = 7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (A) Legumain mRNA expression relative to the geometric mean of CT values of four housekeeping controls in kidney, liver, and spleen (2−∆∆CT; n = 5). (B) One representative immunoblot of legumain and GAPDH in kidney, liver, and spleen (n = 3). (C) Quantifi- cation of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH (housekeeping) in kidney, liver, and spleen from immunoblots represented in (C) (n = 3). (D) Legumain activity (dF/s) in kidney, liver, and spleen adjusted for total protein concentration (µg/mL, n = 5). (E) Legumain plasma concentration (ng/mL) measured by ELISA (n = 5). (F) Cor- relation between legumain (ng/mL and 1,25(OH)2D3 (pmol/L) concentrations in plasma (n = 5). (A,C,E) Two-tailed unpaired Student’s t-test. (D) Mann–Whitney test. Data represent mean ± SEM. * p < 0.05. (F) Simple linear regression. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Activity Assay, Control, Injection, Expressing, Western Blot, Protein Concentration, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 3. Legumain is required for VDBP processing and regulation. (A) Purified VDBP from human plasma (1.9 µM) was incubated in legumain assay buffer (pH 5.8) at 37 ◦C with or without purified active bovine legumain (2 µM) for 5 h before gel electrophoresis and immunoblotting of VDBP (n = 1). (B–H) Wild-type (Lgmn+/+) and legumain-deficient (Lgmn−/−) mice were treated with 50 µg/kg 25(OH)D3 (n = 6–7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (B) One representative immunoblot of VDBP and GAPDH (housekeeping) in kidney and liver (n = 4). (C–F) Quantification of VDBP immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in (B) (n = 4). (C) Hepatic VDBP 45 kDa immunoband. (D) Renal VDBP 45 kDa immunoband. (E) Hepatic VDBP 55 kDa immunoband. (F) Renal VDBP 55 kDa immunoband. (G) Plasma VDBP concentration (µg/mL) was measured by ELISA (n = 6–7). (H) Hepatic VDBP mRNA expression relative to the geometric mean of CT values of four house- keeping controls (2−∆∆CT, n = 5). (C–H) Data represent mean ± SEM. Two-way ANOVA. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. different genotype, same treatment. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Purification, Clinical Proteomics, Incubation, Nucleic Acid Electrophoresis, Western Blot, Control, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 5. Graphical representation of the suggested interplay between vitamin D and legumain. Left panel: Vitamin D (VD3) promotes legumain expression and activity through transcriptional upregulation of the legumain gene (LGMN). The free fraction of circulating VD3 metabolites diffuse through plasma membranes. 25-hydroxyvitamin D (25(OH)D3) is hydroxylated by 1α-hydroxylase (CYP27B1), forming the active metabolite 1α,25-dihydroxyvitamin D (1,25(OH)2D3). 1,25(OH)2D3 binds to the nuclear vitamin D receptor (VDR) and promotes transcription of legumain (LGMN). Synthesized prolegumain is either sorted and activated in the endolysosomal system or released to the extracellular environment. Right panel: In the proximal tubular epithelium, 25(OH)D3 bound to vitamin D binding protein (VDBP) is internalized from the tubular lumen through a megalin/cubilin- mediated process. The vitamin D metabolite is released, enabling subsequent hydroxylation by 1α-hydroxylase (CYP27B1) or 24-hydroxylase (CYP24A1), and VDBP is cleaved by legumain in the endolysosomal system. VDBP cleavage by legumain might be important in controlling the systemic level of vitamin D metabolites. Created with BioRender.com (accessed on 11 December 2023).

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Synthesized, Binding Assay

Journal: International Journal of Biological Sciences

Article Title: miR-487b-3p Suppresses the Proliferation and Differentiation of Myoblasts by Targeting IRS1 in Skeletal Muscle Myogenesis

doi: 10.7150/ijbs.25052

Figure Lengend Snippet: Primer information for miRNA and mRNA quantitative reverse transcription

Article Snippet: Then, the membranes were blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies against Myf5 (MW: 28 kDa; Abscience; USA; 1:1000 dilution), Mef2c (MW: 51 kDa; Abscience; USA; 1:1000 dilution), MyoG (MW: 25 kDa; Abscience; USA; 1:1000 dilution), MyoD (MW: 35 kDa; Abscience; USA; 1:1000 dilution), Pax7 (MW: 57 kDa; EnoGene; China; 1:1000 dilution), PCNA (MW: 36 kDa; EnoGene; China; 1:1000 dilution), IRS1 (MW:130 kDa; Abscience; USA; 1:1000 dilution) and β-tubulin (MW: 55 kDa; Abscience; USA; 1:1000 dilution).

Techniques: Sequencing

Journal: International Journal of Biological Sciences

Article Title: miR-487b-3p Suppresses the Proliferation and Differentiation of Myoblasts by Targeting IRS1 in Skeletal Muscle Myogenesis

doi: 10.7150/ijbs.25052

Figure Lengend Snippet: miR-487b-3p suppresses C2C12 myoblast proliferation. (A, B) MyoD, Pax7, and PCNA mRNA expression after transfection with miR-487b-3p mimics (Mimics), negative control (NC), 2'-O-methylated oligonucleotides against miR-487b-3p (Inhibitors), and single-stranded negative control (Anti-NC) in GM at 24 h and 48 h. (C-F) MyoD (35 kDa; 1:1000 dilution), Pax7 (57 kDa; 1:1000 dilution), and PCNA (36 kDa; 1:1000 dilution) protein expression after transfection with miR-487b-3p mimics, NC, Inhibitors and Anti-NC in GM at 24 h (C, D) and 48 h (E, F). β-tubulin (55 kDa; 1:1000 dilution) was used as an internal control. (G) CCK-8 assay of C2C12 myoblasts at 24 h and 48 h after transfection with miR-487b-3p Mimics, NC, miR-487b-3p Inhibitors and Anti-NC. (H) Representative images of EdU assay of C2C12 myoblasts at 24 h after transfection with Mimics, NC, Inhibitors, Anti-NC in GM. Bars, 100 μm. (I) Quantification of EdU-positive cells(n=6). The results are shown as the mean ± SEM of three independent replicates. One-way ANOVA and t-tests were used for statistical analysis. Asterisks indicate significant differences. * P <0.05, ** P <0.01.

Article Snippet: Then, the membranes were blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies against Myf5 (MW: 28 kDa; Abscience; USA; 1:1000 dilution), Mef2c (MW: 51 kDa; Abscience; USA; 1:1000 dilution), MyoG (MW: 25 kDa; Abscience; USA; 1:1000 dilution), MyoD (MW: 35 kDa; Abscience; USA; 1:1000 dilution), Pax7 (MW: 57 kDa; EnoGene; China; 1:1000 dilution), PCNA (MW: 36 kDa; EnoGene; China; 1:1000 dilution), IRS1 (MW:130 kDa; Abscience; USA; 1:1000 dilution) and β-tubulin (MW: 55 kDa; Abscience; USA; 1:1000 dilution).

Techniques: Expressing, Transfection, Negative Control, Methylation, CCK-8 Assay, EdU Assay

Journal: International Journal of Biological Sciences

Article Title: miR-487b-3p Suppresses the Proliferation and Differentiation of Myoblasts by Targeting IRS1 in Skeletal Muscle Myogenesis

doi: 10.7150/ijbs.25052

Figure Lengend Snippet: Ectopic miR-487b-3p expression inhibits proliferation and differentiation in C2C12 myoblasts. (A) Myf5, MyoG, and Mef2c expression at D1, D3, D5, and D7 after transfection with pcDNA3.1 (+)-miR-487b-3p determined by RT-qPCR in DM. (B, C) Myf5 (28 kDa; 1:1000 dilution), MyoG (25 kDa; 1:1000 dilution) and Mef2c (51 kDa; 1:1000 dilution) protein levels were measured following overexpression of miR-487b-3p with pcDNA3.1(+)-miR-487b-3p in DM at D1, D3, D5 and D7. β-tubulin (55 kDa; 1:1000 dilution) was used as an internal control. (D) MyoD, Pax7, and PCNA mRNA levels after transfection with pcDNA3.1 (+)-miR-487b-3p in GM at 24 h and 48 h. (E) Relative MyoD, Pax7, and PCNA protein expression after transfection with pcDNA3.1 (+)-miR-487b-3p in GM at 24 h and 48 h. (F, G) MyoD (35 kDa; 1:1000 dilution), Pax7 (57 kDa; 1:1000 dilution), and PCNA (36 kDa; 1:1000 dilution) protein expression after transfection with pcDNA3.1 (+)-miR-487b-3p in GM at 24 h and 48 h. β-tubulin (55 kDa; 1:1000 dilution) was used as an internal control. (H) Representative images of EdU assays of C2C12 myoblasts at 24 h after transfection with pcDNA3.1 (+)-miR-487b-3p. Bars, 100 μm. (I) Quantification of EdU-positive cells (n=6). D1, 3, 5, and 7 indicate DM for 1, 3, 5, and 7 days, respectively. The results are shown as the mean ± SEM of three independent replicates. One-way ANOVA and t-tests were used for statistical analysis. Asterisks indicate significant differences. * P <0.05, ** P <0.01.

Article Snippet: Then, the membranes were blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies against Myf5 (MW: 28 kDa; Abscience; USA; 1:1000 dilution), Mef2c (MW: 51 kDa; Abscience; USA; 1:1000 dilution), MyoG (MW: 25 kDa; Abscience; USA; 1:1000 dilution), MyoD (MW: 35 kDa; Abscience; USA; 1:1000 dilution), Pax7 (MW: 57 kDa; EnoGene; China; 1:1000 dilution), PCNA (MW: 36 kDa; EnoGene; China; 1:1000 dilution), IRS1 (MW:130 kDa; Abscience; USA; 1:1000 dilution) and β-tubulin (MW: 55 kDa; Abscience; USA; 1:1000 dilution).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Over Expression

Journal: International Journal of Biological Sciences

Article Title: miR-487b-3p Suppresses the Proliferation and Differentiation of Myoblasts by Targeting IRS1 in Skeletal Muscle Myogenesis

doi: 10.7150/ijbs.25052

Figure Lengend Snippet: IRS1 knockdown inhibits proliferation and differentiation in cultured C2C12 myoblasts. (A) IRS1 expression was measured after transfection with siRNA control (si-NC), Lipo (lipofectamine 2000), si-IRS1-1, si-IRS1-2 and si-IRS1-3 in GM at 24 h. (B, C) IRS1 (130 kDa; 1:1000 dilution) protein expression was examined after transfection with siRNA control (si-NC), si-IRS1-3 in GM at 24 h, 48 h and 72 h. (D, E) MyoD (35 kDa; 1:1000 dilution), Pax7 (57 kDa; 1:1000 dilution), and PCNA (36 kDa; 1:1000 dilution) protein expression was examined after transfection with siRNA control (si-NC), si-IRS1-3 in GM at 24 h (D) and 48 h (E), respectively. (F, G) Myf5 (28 kDa; 1:1000 dilution), MyoG (25 kDa; 1:1000 dilution), and Mef2c (51 kDa; 1:1000 dilution) protein expression was examined after transfection with siRNA control (si-NC), si-IRS1-3 in DM at D2 (F) and D3 (G). β-tubulin (55 kDa; 1:1000 dilution) was used as an internal control. D2, 3 indicate DM for 2 and 3 days. (H) Representative images of EdU assays of C2C12 myoblasts at 24 h after transfection with siRNA control (si-NC), si-IRS1-3. Bars, 100 μm. (I) Quantification of EdU-positive cells (n=6). The results are shown as the mean ± SEM of three independent replicates. One-way ANOVA and t-tests were used for statistical analysis. Asterisks indicate significant differences. * P <0.05, ** P <0.01

Article Snippet: Then, the membranes were blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies against Myf5 (MW: 28 kDa; Abscience; USA; 1:1000 dilution), Mef2c (MW: 51 kDa; Abscience; USA; 1:1000 dilution), MyoG (MW: 25 kDa; Abscience; USA; 1:1000 dilution), MyoD (MW: 35 kDa; Abscience; USA; 1:1000 dilution), Pax7 (MW: 57 kDa; EnoGene; China; 1:1000 dilution), PCNA (MW: 36 kDa; EnoGene; China; 1:1000 dilution), IRS1 (MW:130 kDa; Abscience; USA; 1:1000 dilution) and β-tubulin (MW: 55 kDa; Abscience; USA; 1:1000 dilution).

Techniques: Cell Culture, Expressing, Transfection