Journal: Frontiers in Veterinary Science

Article Title: Screening of Genes Related to Growth, Development and Meat Quality of Sahan Crossbred F1 Sheep Based on RNA-Seq Technology

doi: 10.3389/fvets.2022.831519

Figure Lengend Snippet: Validation of target genes and proteins of longissimus dorsi muscle of STH sheep population and STH×SFK sheep population. (A) The mRNA expression levels of 4 DEGs ( MYL2, PPARD, MSTN and IFRD1 ) were detected by RT-PCR. *Represents P < 0.05, **represents P < 0.01. (B) Two significantly different proteins (IFRD1 and MYL2) were detected by Western blotting. (C) Analyze the integrated optical density values of IFRD1 and MYL2 by Graphpad Prism software. *Represents P < 0.05.

Article Snippet: Five percent skimmed milk powder was used for blocking with an incubation for 2 h; the mouse IFRD1 primary antibody (Santa Cruz, USA, dilution ratio 1:800), MYL2 primary antibody (Santa Cruz, USA, dilution ratio 1:500), and β-actin primary antibody (Bioss, Beijing, China, dilution ratio 1:4000) were added and incubation was performed overnight at 4°C, following which the membranes were washed with PBST; goat anti-mouse IgG/HRP secondary antibody (Bioss, Beijing, China, 1:5000) was added, and incubation was performed at 37°C for 2 h. Thereafter, PBST was used in washing steps and ECL was used to for color development for further analysis. β -actin expression was considered as a control.

Techniques: Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Software

Journal: Frontiers in Veterinary Science

Article Title: Screening of Genes Related to Growth, Development and Meat Quality of Sahan Crossbred F1 Sheep Based on RNA-Seq Technology

doi: 10.3389/fvets.2022.831519

Figure Lengend Snippet: Expression location of MYL2 and IFRD1 protein in STH and STH×SFK longissimus dorsi muscle (IF×400). CYT, Cytoplasm; N, Nucleus; CT, Connective tissue.

Article Snippet: Five percent skimmed milk powder was used for blocking with an incubation for 2 h; the mouse IFRD1 primary antibody (Santa Cruz, USA, dilution ratio 1:800), MYL2 primary antibody (Santa Cruz, USA, dilution ratio 1:500), and β-actin primary antibody (Bioss, Beijing, China, dilution ratio 1:4000) were added and incubation was performed overnight at 4°C, following which the membranes were washed with PBST; goat anti-mouse IgG/HRP secondary antibody (Bioss, Beijing, China, 1:5000) was added, and incubation was performed at 37°C for 2 h. Thereafter, PBST was used in washing steps and ECL was used to for color development for further analysis. β -actin expression was considered as a control.

Techniques: Expressing

Journal: Biomedicines

Article Title: Gastrodin Mitigates Ketamine-Induced Inhibition of F-Actin Remodeling and Cell Migration by Regulating the Rho Signaling Pathway

doi: 10.3390/biomedicines13030649

Figure Lengend Snippet: The effects of ketamine and gastrodin on the expression and activation of ROCK1 and MLC2. Protein extracts collected from ( a ) B35 and ( b ) C6 cells treated with ketamine and/or gastrodin were examined for the expression of ROCK1, MLC2, and p-MLC2. The bar charts ( c , d ) of the quantitative results were generated from triplicate Western blot data from three different batches of drug-treated cells. A p -value less than 0.01 (**) or 0.05 (*) from ANOVA followed by Dunnett’s test was considered significant. CTRL, control; ketamine, KET; gastrodin, GAS.

Article Snippet: Primary antibodies used included those for beta-actin (GTX26276, GeneTex Inc., Irvine, CA, USA), RhoGDI1 (#2564, Cell Signaling Technology, Danvers, MA, USA), phosphorylated RhoGDI1 (phospho S174, ab74142, Abcam, Cambridge, UK), RhoA (#2117, Cell Signaling Technology), anti-CDC42 (#2462, Cell Signaling Technology), CDC42 (#2462, Cell Signaling Technology), Rac1 (GTX100761, GeneTex Inc.), phosphorylated RhoA Ser188 (sc-32954, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated CDC42 (ab74142, Abcam, Cambridge, UK), phosphorylated Rac1 (phospho S71) (PAB7743, Abnova Corporation, Taipei, Taiwan), ROCK1 (ab45171, Abcam), myosin light chain 2 (#3672, Cell Signaling Technology), phosphorylated MLC2 (Ser-18) (TA309976, OriGene Technologies, Rockville, MD, USA), cofilin-1(H00001072-M04, Abnova Corporation, Taipei, Taiwan), and profilin-1(#3237, Cell Signaling Technology).

Techniques: Expressing, Activation Assay, Generated, Western Blot, Control

Journal: Biomedicines

Article Title: Gastrodin Mitigates Ketamine-Induced Inhibition of F-Actin Remodeling and Cell Migration by Regulating the Rho Signaling Pathway

doi: 10.3390/biomedicines13030649

Figure Lengend Snippet: The effects of ketamine and gastrodin on the regulation of ROCK1, MLC2, CFL1, and PFN1 in the PFC of SD rat. Protein extracts collected from the PFC of SD rats treated with ketamine and/or gastrodin were examined for the regulation of ( a ) ROCK1 and PFN1 and ( b ) MLC2 and FCL1. The bar charts ( c , d ) of the quantitative results were generated from triplicate Western blot data from three drug-treated rats. A p -value less than 0.01 (**) or 0.05 (*) from ANOVA followed by Dunnett’s test was considered significant. CTRL, control; ketamine, KET; gastrodin, GAS.

Article Snippet: Primary antibodies used included those for beta-actin (GTX26276, GeneTex Inc., Irvine, CA, USA), RhoGDI1 (#2564, Cell Signaling Technology, Danvers, MA, USA), phosphorylated RhoGDI1 (phospho S174, ab74142, Abcam, Cambridge, UK), RhoA (#2117, Cell Signaling Technology), anti-CDC42 (#2462, Cell Signaling Technology), CDC42 (#2462, Cell Signaling Technology), Rac1 (GTX100761, GeneTex Inc.), phosphorylated RhoA Ser188 (sc-32954, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated CDC42 (ab74142, Abcam, Cambridge, UK), phosphorylated Rac1 (phospho S71) (PAB7743, Abnova Corporation, Taipei, Taiwan), ROCK1 (ab45171, Abcam), myosin light chain 2 (#3672, Cell Signaling Technology), phosphorylated MLC2 (Ser-18) (TA309976, OriGene Technologies, Rockville, MD, USA), cofilin-1(H00001072-M04, Abnova Corporation, Taipei, Taiwan), and profilin-1(#3237, Cell Signaling Technology).

Techniques: Generated, Western Blot, Control

Journal: Scientific reports

Article Title: Identification of MYLK3 mutations in familial dilated cardiomyopathy.

doi: 10.1038/s41598-017-17769-1

Figure Lengend Snippet: Figure 4. Western blot analysis suggested decreased expression levels of mutant cMLCK and MLC2 phosphorylation. (a) Expression of wild-type and mutant cMLCK protein in transfected HEK293T cells. Western blot analysis showed the reduced expression of mutant cMLCK protein. The blots were run under the same experimental conditions. The uncropped images are in Supplementary Fig. 2. (b) Densitometric quantification of western blots. Data are expressed as mean ± SEM (N = 3). *P < 0.05. (c) Wild-type or mutant MYLK3 transfected HEK293T cells were treated with cycloheximide (CHX) for indicated times. Western blot analysis showed rapid reduction of mutant proteins. Data are expressed as mean ± SEM (N = 3). *P < 0.05. The blots were run under the same experimental conditions. The uncropped images are in Supplementary Fig. 3. (d) Phos-tag SDS-PAGE followed by western blot analysis showed phosphorylated and non-phosphorylated forms of MLC2 using anti-FLAG antibody. Levels of phosphorylated MLC2 were remarkably reduced when co-transfected with mutant MYLK3 and MYL2 constructs. The ratio of phosphorylated MLC2 to total MLC2 (phosphorylated MLC2+ non-phosphorylated MLC2) was determined. Data are expressed as mean ± SEM (N = 3). *P < 0.05. The uncropped images are in Supplementary Fig. 4.

Article Snippet: A full-length cDNA clone of human MYL2 (NM_000432) with an C-terminal Myc-FLAG-Tag was obtained from Origene (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Mutagenesis, Phospho-proteomics, Transfection, SDS Page, Construct