Journal: International Journal of Biological Sciences

Article Title: Proton-sensing GPCR-YAP Signalling Promotes Cell Proliferation and Survival

doi: 10.7150/ijbs.12500

Figure Lengend Snippet: Regulation of GPCR-Rho signalling on YAP activities (A) Low extracellular pH activates ROCK kinase while inhibits LATS kinase. The activity of ROCK kinase was monitored by phosphorylation of MYPT1, a substrate of ROCK kinase. Phosphorylation of LATS1 at T1079 and S909 which results in inhibition of LATS kinase was repressed by low extracellular pH. (B) Inhibition of Rho signalling interferes with dephosphorylation of YAP in response to decreased extracellular pH. ECC-1 cells were cultured at high cell density and then treated with C3 (3μg/mL) and Y27632 (50mM) in medium with low pH (pH=7.0) for 3 hours. (C) Nuclear localization of YAP induced by low extracellular pH is blocked by inhibition of Rho signalling. BMSCs were cultured at high cell density and then treated with C3 (3μg/mL) and Y27632 (50mM) at pH=7.0 for 3 hours. (D) Knockdown of GPR68 blocks pH-dependent regulation on YAP. BMSCs transfected with control siRNA or GPR68 siRNA and cultured at pH=7.0 for 3 hours. (E) ACs were transfected with empty vector or GPR68 vector and cultured at pH=7.0 for 3 hours. C3 (3μg/mL) and Y27632 (50mM) were treated as indicated. All experiments were performed at least 3 times.

Article Snippet: YAP (#14074), p-S127 (#13008), ROCK1 (#4035), LATS1 (#3477), p-T1079 (#8654), p-S909 (#9157), MYPT1 (#5143), p-T853 (#4563) and GAPDH (#5174) antibodies were obtained from Cell Signaling Technology.

Techniques: Activity Assay, Phospho-proteomics, Inhibition, De-Phosphorylation Assay, Cell Culture, Knockdown, Transfection, Control, Plasmid Preparation

Journal: Circulation

Article Title: Pivotal Role of Rho-Associated Kinase 2 in Generating the Intrinsic Circadian Rhythm of Vascular Contractility

doi: 10.1161/circulationaha.112.135608

Figure Lengend Snippet: Figure 4. The circadian changes in the activity and the expression of Rho-asso ciated kinase 2 (ROCK2) in porcine coro nary artery smooth muscle cells. A, The circadian changes in the level of MYPT1 phosphorylation at residues correspond ing with Thr853 (n=5) and Thr696 (n=4) in human MYPT1 obtained 2 minutes after the stimulation with 1 U/mL of thrombin. B, The representative immunoblot detec tion of ROCK2 and summaries of circadian changes in the level of ROCK2 protein (left; n=5) and mRNA (right; n=4). C, The circa dian patterns of the protein levels of MLC kinase (MLCK), protein kinase C (PKC)-∝, and Zipper-interacting kinase (ZIPK; n=3). D, The circadian pattern of the activity of RhoA as evaluated by the level of the GTP-bound form of RhoA in the total RhoA (GTP-RhoA/total RhoA) obtained 2 min utes after the stimulation with 1 U/mL of thrombin (n=4). The values of GTP-RhoA/ total RhoA were obtained after normalizing the levels of GTP-RhoA and total RhoA to those of the pull down probe and actin, respectively. The data are expressed as the relative values to those obtained at 48 hours (A and B) and 24 hours (C and D). All of the data are expressed as the mean±SEM; #P<0.05 vs 48 hours (Steel test; A and B, left). ##P<0.01 (Student t test; B, right).

Article Snippet: The primary antibodies used were generated against MLC (sc-15370; Santa Cruz, Santa Cruz, CA, U.S.A.), phosphor-MLC at Ser19 (#3671; Cell signaling, Beverly, MA, U.S.A.), phosphor-MLC at Thr18 and Ser19 (#3674; Cell signaling), MYPT1 (612164; BD Transduction, San Jose, CA, U.S.A.), phosphor-MYPT1 at Thr696 (#07-251; Upstate; Lake Placid, NY, U.S.A.), phosphor-MYPT1 at Thr853 (#4563; Cell signaling), ROCK2 (610624; BD Transduction), ROCK1 (#4035; Cell signaling), MLCK (ab76092; Abcam, Cambridge, UK), ZIPK (#2928; Cell signaling), PKCα (sc-8393; Santa Cruz), RORα (sc-28612; Santa Cruz) and RORγ (sc-28559; Santa Cruz).

Techniques: Activity Assay, Expressing, Phospho-proteomics, Western Blot

Journal: Placenta

Article Title: Differential expression of PPP1R12A transcripts, including those harbouring alternatively spliced micro-exons, in placentae from complicated pregnancies.

doi: 10.1016/j.placenta.2024.04.005

Figure Lengend Snippet: Fig. 1. Schematic overview of the PPP1R12A locus on chromosome 12. (A) The entire regions showing the orientation of the PPP1R12A and PPP1R12A-AS1 transcripts, as well as the promoter CpG island (green). The insert is a focal view point of intron 23 showing the position of the micro-exons and read-through transcripts. Arrows indicate the direction of transcription. (B) Sanger sequencing of isoform-specific RT-PCR amplicons incorporating the LZ-micro-exon or read- through sequences. (C) Bar graphs showing the relevant PPP1R12A isoform abundance in paired placenta vessel and villous samples, as well as uterus.

Article Snippet: Afterward, the electrophoretic transfer of proteins onto polyvinylidene fluoride membranes was performed, with efficient transfer confirmed using Ponceau S staining, followed by probing with antibodies against ACTB (α-Actin-HRP Sigma #A3854, 1:10,000 dilution), PPP1R12A (α-MYPT1 ProteinTech #22117-1-AP, 1:10,000 dilution) and secondary antibody conjugated with horseradish peroxidase (α-rabbit-IgG-HRP, Sigma #A0545,1:200 dilution).

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: Placenta

Article Title: Differential expression of PPP1R12A transcripts, including those harbouring alternatively spliced micro-exons, in placentae from complicated pregnancies.

doi: 10.1016/j.placenta.2024.04.005

Figure Lengend Snippet: Fig. 2. Expression profiling of the PPP1R12A transcript isoforms in placenta biopsies from complicated pregnancies. Quantification of expression levels of (A) WT, (B) all isoforms, (C) 4 bp micro-exon, (D) the read-through, (E) 31 bp micro-exon, (F) the 13 bp micro-exon and (G) the PPP1R12A-AS1 transcript in control, IUGR and PE samples (n = 18 per group) by qRT-PCR. The results are presented as box plots, with individual data points (black dots) shown. All expression levels were normalized to the average of ACTB and RPL19 housekeeping genes. To determine the statistical significance of the difference between the IUGR/PE and control groups, Wilcoxon signed-rank test was used and p values are indicated for each comparison.

Article Snippet: Afterward, the electrophoretic transfer of proteins onto polyvinylidene fluoride membranes was performed, with efficient transfer confirmed using Ponceau S staining, followed by probing with antibodies against ACTB (α-Actin-HRP Sigma #A3854, 1:10,000 dilution), PPP1R12A (α-MYPT1 ProteinTech #22117-1-AP, 1:10,000 dilution) and secondary antibody conjugated with horseradish peroxidase (α-rabbit-IgG-HRP, Sigma #A0545,1:200 dilution).

Techniques: Expressing, Control, Quantitative RT-PCR, Comparison

Journal: Placenta

Article Title: Differential expression of PPP1R12A transcripts, including those harbouring alternatively spliced micro-exons, in placentae from complicated pregnancies.

doi: 10.1016/j.placenta.2024.04.005

Figure Lengend Snippet: Fig. 3. Epigenetic analysis of the PPPIR12A promoter interval. (A) Schematic overview of the promoter interval showing the location of the CpG island and primers used for ChIP and bisulphite PCR. (B) Characterization of the promoter CpG islands in uterus, blood vessel and placenta-derived DNA samples. The Sanger sequence tracks show the location of selected unmethylated CpG positions before cloning of the PCR amplicons. The entire DNA methylation profile is shown for the placenta PCR product following cloning of individual DNA strands. Each circle represents a single CpG on a DNA strand. (•) methylated cytosine, (o) unmethylated cytosine. Each row corresponds to an individual cloned sequence. (C) Quantitative PCR on ChIP material. Precipitations were normalized to the SNURF promoter since opposing alleles have active/repressive histone modification profiles. The graphs represent the mean values of triplicate PCRs. (D) Pyrosequencing quantification of the PPP1R12A promoter. The violin plots include the mean (black dots). To determine the statistical significance of the difference between the IUGR/PE and control groups (n = 18 per group), Student’s two-tailed t-test was used, and p values are indicated for each comparison.

Article Snippet: Afterward, the electrophoretic transfer of proteins onto polyvinylidene fluoride membranes was performed, with efficient transfer confirmed using Ponceau S staining, followed by probing with antibodies against ACTB (α-Actin-HRP Sigma #A3854, 1:10,000 dilution), PPP1R12A (α-MYPT1 ProteinTech #22117-1-AP, 1:10,000 dilution) and secondary antibody conjugated with horseradish peroxidase (α-rabbit-IgG-HRP, Sigma #A0545,1:200 dilution).

Techniques: Derivative Assay, Sequencing, Cloning, DNA Methylation Assay, Methylation, Clone Assay, Real-time Polymerase Chain Reaction, Modification, Control, Two Tailed Test, Comparison

Journal: American journal of physiology. Cell physiology

Article Title: Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE.

doi: 10.1152/ajpcell.00327.2015

Figure Lengend Snippet: Fig. 1. Phosphorylation of full-length myosin-targeting subunit 1 of myosin light chain phosphatase (MYPT1) by Rho-associated coiled-coil kinase (ROCK). A: Coo- massie blue-stained SDS gel of purified recombinant MYPT1 (1 and 2 g). M, molecular mass marker (in kDa). B: purified, recombinant full-length MYPT1 was incubated with constitutively active ROCK (see METH- ODS). Reactions were started at time 0 by addition of [-32P]ATP, and samples of reaction mixtures were withdrawn at the indicated times for quantification of 32P incorporation by scintillation counting. Values are means SE (n 4–5). Inset: samples withdrawn at the indicated times were also subjected to Phos-tag SDS- PAGE and Western blotting with anti-MYPT1. 0P, 1P, and 2P indicate MYPT1 bands with 0, 1, or 2 sites phosphorylated, respectively. C: samples withdrawn at the indicated times were subjected to SDS-PAGE and Coomassie blue staining (left) and autoradiography to detect incorporated 32P (pMYPT1; right). M, molecular mass marker. D: samples withdrawn at the indicated times were subjected to Phos-tag SDS-PAGE and West- ern blotting with anti-MYPT1 (pan-MYPT1), anti-phos- phorylated (Thr697) MYPT1 (pT697), and anti-phos- phorylated (Thr855) MYPT1 (pT855).

Article Snippet: The following primary antibodies were used: anti-pan-MYPT1 (1:5,000 dilution for tissue samples or 1:10,000 dilution for purified protein samples), anti-phosphorylated (Ser854/Thr855) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Ser696/Thr697) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Thr697) MYPT1 (1:2,000 dilution), anti-phosphorylated Thr855 MYPT1 (1:5,000 dilution), and anti-phosphorylated (Ser696) and anti-phosphorylated (Ser696/Ser854) MYPT1 (custom-made peptide-directed antibodies, New England Peptide; both at 1:500 dilution).

Techniques: Phospho-proteomics, Staining, SDS-Gel, Recombinant, Marker, Incubation, SDS Page, Western Blot, Autoradiography

Journal: American journal of physiology. Cell physiology

Article Title: Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE.

doi: 10.1152/ajpcell.00327.2015

Figure Lengend Snippet: Fig. 3. Phosphorylation of full-length MYPT1 by a high concentration of PKA. A: purified, recombinant full-length MYPT1 was incubated with the catalytic subunit of PKA (10 g/ml; see METHODS). Reactions were started at time 0 by addition of [-32P]ATP, and samples of reaction mixtures were withdrawn at the indicated times for quantification of 32P incorporation. Values are means SE (n 3–4). Inset: samples withdrawn at the indicated times were subjected to Phos-tag SDS-PAGE and Western blotting with anti-MYPT1. B: samples withdrawn at the indicated times were subjected to SDS-PAGE and Coomassie blue staining (left) and autoradiography to detect incorporated 32P (pMYPT1; right). M, molecular mass marker.

Article Snippet: The following primary antibodies were used: anti-pan-MYPT1 (1:5,000 dilution for tissue samples or 1:10,000 dilution for purified protein samples), anti-phosphorylated (Ser854/Thr855) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Ser696/Thr697) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Thr697) MYPT1 (1:2,000 dilution), anti-phosphorylated Thr855 MYPT1 (1:5,000 dilution), and anti-phosphorylated (Ser696) and anti-phosphorylated (Ser696/Ser854) MYPT1 (custom-made peptide-directed antibodies, New England Peptide; both at 1:500 dilution).

Techniques: Phospho-proteomics, Concentration Assay, Recombinant, Incubation, SDS Page, Western Blot, Staining, Autoradiography, Marker

Journal: American journal of physiology. Cell physiology

Article Title: Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE.

doi: 10.1152/ajpcell.00327.2015

Figure Lengend Snippet: Fig. 2. Phosphorylation of full-length MYPT1 by PKA. A: purified, recombi- nant full-length MYPT1 was incubated with PKA (1 g/ml; see METHODS). Reactions were started at time 0 by addition of [-32P]ATP, and samples of reaction mixtures were withdrawn at the indicated times for quantification of 32P incorporation. Values are means SE (n 3–6). Inset: samples with- drawn at the indicated times were subjected to Phos-tag SDS-PAGE and Western blotting with anti-MYPT1. 0P, 1P, 2P, 3P, and 4P indicate MYPT1 bands with 0, 1, 2, 3, or 4 sites phosphorylated. B: samples withdrawn at the indicated times were subjected to Phos-tag SDS-PAGE and Western blotting with anti-MYPT1 (pan MYPT1), anti-phosphorylated (Thr697) MYPT1 (pT697), anti-phosphorylated (Thr855) MYPT1 (pT855), anti-phosphorylated (Ser696/Thr697) MYPT1 (pS696pT697), and anti-phosphorylated (Ser696/854) MYPT1 (pS696pS854).

Article Snippet: The following primary antibodies were used: anti-pan-MYPT1 (1:5,000 dilution for tissue samples or 1:10,000 dilution for purified protein samples), anti-phosphorylated (Ser854/Thr855) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Ser696/Thr697) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Thr697) MYPT1 (1:2,000 dilution), anti-phosphorylated Thr855 MYPT1 (1:5,000 dilution), and anti-phosphorylated (Ser696) and anti-phosphorylated (Ser696/Ser854) MYPT1 (custom-made peptide-directed antibodies, New England Peptide; both at 1:500 dilution).

Techniques: Phospho-proteomics, Incubation, SDS Page, Western Blot

Journal: American journal of physiology. Cell physiology

Article Title: Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE.

doi: 10.1152/ajpcell.00327.2015

Figure Lengend Snippet: Fig. 5. Phosphorylation of full-length MYPT1 by PKA and ROCK alone, together, and sequentially. A: purified, recombinant full-length MYPT1 was incubated with the catalytic subunit of PKA and/or constitutively active ROCK (see METHODS). Reactions were started at time 0 by addition of [-32P]ATP, and samples of reaction mixtures were withdrawn at the indicated times for quantification of 32P incorporation. At 60.25 min (vertical arrow), buffer or the other kinase was added. Values are means SE (n 5). Œ, PKA ROCK; , PKA then ROCK; , PKA; , ROCK; o, ROCK then PKA. B: samples withdrawn at the indicated times were subjected to Phos-tag SDS-PAGE and Western blotting with anti-MYPT1 (left) and autoradiography to detect incor- porated 32P (right).

Article Snippet: The following primary antibodies were used: anti-pan-MYPT1 (1:5,000 dilution for tissue samples or 1:10,000 dilution for purified protein samples), anti-phosphorylated (Ser854/Thr855) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Ser696/Thr697) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Thr697) MYPT1 (1:2,000 dilution), anti-phosphorylated Thr855 MYPT1 (1:5,000 dilution), and anti-phosphorylated (Ser696) and anti-phosphorylated (Ser696/Ser854) MYPT1 (custom-made peptide-directed antibodies, New England Peptide; both at 1:500 dilution).

Techniques: Phospho-proteomics, Recombinant, Incubation, SDS Page, Western Blot, Autoradiography

Journal: American journal of physiology. Cell physiology

Article Title: Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE.

doi: 10.1152/ajpcell.00327.2015

Figure Lengend Snippet: Fig. 6. Microcystin-induced phosphorylation of 20-kDa regulatory light chain of smooth muscle myosin (LC20) and MYPT1 in skinned rat caudal arterial smooth muscle. A: after control pCa 4.5-induced contractions (the last of which is shown) and relaxation at pCa 9, Triton-skinned rat caudal arterial smooth muscle strips were treated with 1 M microcystin at pCa 9 and force was recorded continuously. B and C: skinned smooth muscle strips incubated in the presence (pCa 4.5) or absence (pCa 9) of Ca2 or treated with 1 M microcystin (MC) at pCa 9 for the indicated times (arrows in A) were subjected to Phos-tag SDS-PAGE and Western blotting (IB) with anti-LC20 or anti-MYPT1. Unphosphorylated, monophosphorylated, and diphosphory- lated LC20 species are labeled 0P-LC20, 1P-LC20, and 2P-LC20, respectively. Unphosphorylated, recombinant full-length MYPT1 (0P-MYPT1) indicates migration of the unphosphorylated protein for comparison. Maximally phos- phorylated MYPT1 is also labeled (maxP-MYPT1). D: Triton-skinned rat caudal arterial smooth muscle strips were incubated in the absence of Ca2 (pCa 9) or treated with microcystin at pCa 9 for the indicated times prior to Phos- tag SDS-PAGE in the presence of MnCl2 or EDTA and Western blotting with anti-MYPT1. Data are representative of 5 (B), 3 (C), and 4 (D) independent experiments.

Article Snippet: The following primary antibodies were used: anti-pan-MYPT1 (1:5,000 dilution for tissue samples or 1:10,000 dilution for purified protein samples), anti-phosphorylated (Ser854/Thr855) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Ser696/Thr697) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Thr697) MYPT1 (1:2,000 dilution), anti-phosphorylated Thr855 MYPT1 (1:5,000 dilution), and anti-phosphorylated (Ser696) and anti-phosphorylated (Ser696/Ser854) MYPT1 (custom-made peptide-directed antibodies, New England Peptide; both at 1:500 dilution).

Techniques: Phospho-proteomics, Control, Incubation, SDS Page, Western Blot, Labeling, Recombinant, Migration, Comparison

Journal: American journal of physiology. Cell physiology

Article Title: Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE.

doi: 10.1152/ajpcell.00327.2015

Figure Lengend Snippet: Fig. 7. Calyculin A-induced phosphorylation of LC20 and MYPT1 in intact rat caudal arterial smooth muscle. A: after control KCl (membrane depolarization)-induced contrac- tions (the last of which is shown) and relaxation in HEPES- Tyrode (H-T) buffer, intact rat caudal arterial smooth mus- cle strips were treated with 0.5 M calyculin A in the presence of Ca2 and force was recorded continuously. B and C: intact smooth muscle strips incubated in the absence (Ca2) or presence (Ca2) of Ca2 or treated with 0.5 M calyculin A in the presence of Ca2 for the indicated times were subjected to Phos-tag SDS-PAGE and Western blotting with anti-LC20 or anti-MYPT1. Unphosphorylated, monophosphorylated, and diphosphorylated LC20 species are labeled 0P-LC20, 1P-LC20, and 2P-LC20, respectively. Unphosphorylated, recombinant full-length MYPT1 (0P- MYPT1) indicates migration of the unphosphorylated pro- tein for comparison. Maximally phosphorylated MYPT1 is also labeled (maxP-MYPT1). D: Phos-tag SDS-PAGE of Triton-skinned rat caudal arterial smooth muscle strips treated for 60 min with microcystin (1 M) or intact strips treated for 60 min with calyculin A (0.5 M) and subjected to Western blotting with anti-MYPT1. Purified, unphos- phorylated, recombinant full-length MYPT1 (MYPT1) is included for comparison. Data are representative of 5 inde- pendent experiments. E: intact smooth muscle strips incu- bated with 0.5 M calyculin A in the presence of Ca2 for the indicated times were subjected to Phos-tag SDS-PAGE and Western blotting with anti-MYPT1 (pan-MYPT1), anti- phosphorylated (Thr697) MYPT1 (pT697), anti-phosphory- lated (Thr855) MYPT1 (pT855), anti-phosphorylated (Ser696/Thr697) MYPT1 (pS696pT697), and anti-phosphor- ylated (Ser854/Thr855) MYPT1 (pS854pT855). Horizontal bars at left of Western blots for pan-MYPT1, pT697, and pT855 indicate that the major band recognized by the pan-MYPT1 antibody at time 0 corresponds to the major band recognized by pT697 and to a minor band recognized by pT855, as confirmed by reprobing with the complemen- tary antibody. Data are representative of 3 independent experiments.

Article Snippet: The following primary antibodies were used: anti-pan-MYPT1 (1:5,000 dilution for tissue samples or 1:10,000 dilution for purified protein samples), anti-phosphorylated (Ser854/Thr855) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Ser696/Thr697) MYPT1 (custom-made peptide-directed antibody; 1:1,000 dilution), anti-phosphorylated (Thr697) MYPT1 (1:2,000 dilution), anti-phosphorylated Thr855 MYPT1 (1:5,000 dilution), and anti-phosphorylated (Ser696) and anti-phosphorylated (Ser696/Ser854) MYPT1 (custom-made peptide-directed antibodies, New England Peptide; both at 1:500 dilution).

Techniques: Phospho-proteomics, Control, Membrane, Incubation, SDS Page, Western Blot, Labeling, Recombinant, Migration, Comparison

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: TGF-β1 Evokes Human Airway Smooth Muscle Cell Shortening and Hyperresponsiveness via Smad3

doi: 10.1165/rcmb.2017-0247OC

Figure Lengend Snippet: Effect of Rho kinase (ROCK) inhibition on TGF-β1–induced MLC20 phosphorylation and hyperresponsiveness in HASM cells. (A) HASM cells were treated with TGF-β1 (10 ng/mL) for 18 hours and then stimulated with Cch (10 μM) or Hist (1 μM) for 10 minutes (n = 5 ± SEM). (B) HASM cells were pretreated with the ROCK inhibitor Y-27632 for 15 minutes. HASM cells were then treated with TGF-β1 (10 ng/mL) for 18 hours and stimulated with Cch or Hist (n = 8 ± SEM, n = 4 ± SEM). pMYPT1/MYPT1 vehicle versus Y-27632 immunoblots show two different experiments representative of multiple donor observations. pMLC/MLC vehicle versus Y-27632 immunoblots show one representative immunoblot from multiple donor observations. (C) HASM cells were transfected with siRNA targeted against RhoA or a nontargeting (NT) siRNA pool, and then treated with TGF-β1 (10 ng/mL, 18 h) and stimulated with Cch (20 μM, 10 min; n = 3 ± SEM). *P < 0.05; **P < 0.01; ***P < 0.001. MYPT1 = myosin phosphatase target subunit 1; pMYTP1 = phosphorylated myosin phosphatase target subunit 1.

Article Snippet: Antibodies were purchased from Cell Signaling Technologies (pMLC [3674S], pMYPT1-s507 [3040S], GAPDH [2118S], and RhoA [2117S]), Abcam (Smad3 [ab28379], Smad2 [ab71109], and pSmad3 [ab52903]), EMT Millipore (MLC [MABT180]), BD Biosciences (total MYPT1 [612165]), and Santa Cruz Biotechnology (Smad4 [sc-7966]). siRNA was purchased from ThermoFisher Scientific (Smad3 [VHS41114]), Dharmacon (Smad2 [L-003561-00]), and Qiagen (RhoA [S102654211]).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Transfection

Journal: Bioscience Reports

Article Title: Excessive hypercholesterolemia in pregnancy impairs the cardiovascular health of the adult female and male offspring

doi: 10.1042/BSR20253861

Figure Lengend Snippet: Representative blots ( A ) and densitometry of TP receptor (B; normalized to total protein), ROCK1 (C; normalized to total protein), ROCK2 (D; normalized to total protein), pMYPT1 (E; normalized to MYPT1), and MYPT1 (F; normalized to total protein) in thoracic aortas from the adult female offspring born from CD (black symbols) or HCD (orange symbols) pregnancies. Data are presented as means±SEM and were analyzed with an unpaired Student’s t-test ( n =6/group; one offspring/dam/group). * P <0.05. Abbreviation: SEM, standard error of the mean.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies for the TP receptor (1:1,000, rabbit polyclonal; Cayman Chemical, Ann Arbor, MI, U.S.A.), ROCK1 (1:5,000, rabbit polyclonal, 21850–1-AP; Proteintech, Rosemont, IL, U.S.A.), ROCK2 (1:4,000, rabbit polyclonal, 21645–1-AP; Proteintech), pMYPT1 Thr855 (1:250, rabbit polyclonal, 4563; New England Biolabs, Whitby, ON, Canada), or MYPT1 (1:3,000, mouse monoclonal, 66506–1-lg; Proteintech).

Techniques: