Journal: Circulation

Article Title: Detection of Cardiac Allograft Rejection and Response to Immunosuppressive Therapy With Peripheral Blood Gene Expression

doi: 10.1161/01.cir.0000150539.72783.bf

Figure Lengend Snippet: Figure 4. We quantified transcript abundance of 2 candidate markers, CFLAR and SOD2, using qRT-PCR. Data are displayed as fold changes in expression in rejection (n10) and postrejec- tion (n8), each compared with control (n5). In agreement with microarray findings, both CFLAR and SOD2 expression were decreased in rejection. CFLAR expression returned toward control levels in postrejection samples, and SOD2 expression remained low, consistent with persistent partial activation of cir- culating leukocytes after treatment of rejection. *P0.05 com- pared with control by Wilcoxon rank-sum test.

Article Snippet: The specific assays used were Hs00153439_m1 (CFLAR), Hs00167309_m1 (SOD2), and Hs99999905_m1 (GAPDH).

Techniques: Quantitative RT-PCR, Expressing, Control, Microarray, Activation Assay

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Domain arrangement in the Z-disk/I-band region of cardiac titin N2B/N2BA isoforms and in PP5. Constructs generated for yeast-two-hybrid (Y2H) screens marked in red, those for GST-pulldown assays in blue, and N2Bus-binding amino acids (AA) of PP5 in green. Ig’s, immunoglobulin-like domains. Inset: Epitope positions of all phospho-titin antibodies used in this study. (m), anti-mouse; (h), anti-human. b Summary of results of GST-pulldown assays probing interaction of N2Bus with full-length PP5, PP5 catalytic subunit (PP5c), or N-terminal PP5 fragments (T; T+). PP5-binding to PEVK or N2A titin domains was also tested. GST, glutathione S transferase (for negative control). Each test was performed a minimum of two times, mostly three times, with identical results. c Demonstration of PP5-N2Bus association by co-immunoprecipitation assay. PP5 (HA-tag) immunoprecipitations (IP) and whole-cell lysates (WCL) from HEK cells analyzed by western blot for N2Bus (myc-tag) and PP5. PP5 -/+indicates absence/presence of PP5 in the assay. This test was performed three times, with identical results. d Binding of PP5 to sarcomeric I-bands is enhanced by phosphorylation. Top: the experimental design for the stretching of single myofibrils and immunofluorescence image of stretched human cardiac myofibril incubated in relaxing buffer with Cy3-conjugated secondary antibodies alone (control), as well as phase-contrast image (PC). Bottom: representative images of myofibrils incubated with exogenous PP5c and stained against PP5c. The myofibril on the right was incubated with catalytic subunit of PKA before PP5c-treatment (arrowheads, I-band localization of PP5). Binding visualized by anti-PP5c primary and Cy3-conjugated secondary antibodies. Similar results were obtained from four other myofibrils per group. Bars, 2 µm. e Results of GST-pulldown assays probing interaction of PP5 with unphosphorylated N2Bus or N2Bus phosphorylated by PKA/PKG, as well as wildtype (WT) and S4185A mutant of C-terminal N2Bus fragment, both phosphorylated by cGMP-activated PKG. Left: representative immunoblots using anti-PP5 antibody. Right: relative signal intensities in ‘Bound’ lane, normalized to the mean intensity of the respective non-phosphorylated/C-Term WT control. Data are mean ± s.e.m., n = 4 assays/condition. * p < 0.05 and ** p < 0.01, by two-tailed Student’s t -test

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Construct, Generated, Binding Assay, Negative Control, Co-Immunoprecipitation Assay, Western Blot, Phospho-proteomics, Immunofluorescence, Incubation, Control, Staining, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a PP5 expression by western blot in fetal (E18), newborn (P1) and adult rat heart tissue. PP5 indexed to GAPDH. Data are mean ± s.e.m., n = 5; * p < 0.05, by two-tailed Student’s t -test. b PP5 expression by western blot in neonatal rat ventricular myocyte (NRVM) cultures under baseline conditions (control) and following treatment with arachidonic acid (aa; 200 µM, 2 h) or okadaic acid (oa, 10 nM, 1 h). PP5 indexed to GAPDH. Data are mean ± s.e.m., n = 5 (three different cell culture batches); * p < 0.05 and ** p < 0.01, by Bonferroni adjusted t -test. c PP5 localization in control, aa-treated, and oa-treated NRVM cultures by indirect immunofluorescence. PP5 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with α-actinin antibody (secondary antibody: FITC-conjugated IgG). The merged image also shows staining of nuclei using Hoechst. Bars, 10 µm. d PP5 localization in control, aa-treated, and oa-treated adult rat cardiomyocyte (ARC) cultures by indirect immunofluorescence. The same antibodies as in c were used. Bars, 5 µm. Right bar graph shows proportion of ARC exhibiting clear PP5 striations or no such striated pattern, for each group. Numbers above columns indicate total number of cells included in the analysis. e Total titin phosphorylation in the three ARC groups measured by ProQ Diamond phosphoprotein vs. Sypro Ruby total protein stain. Bar graph shows mean ± s.e.m., n = 9 (from three independent cell preparations); * p < 0.05, by two-tailed Student’s t -test. f Localization of phosphoserine P-S3991 (titin N2Bus) in control, aa-treated, and oa-treated ARC cultures by indirect immunofluorescence, using anti-N2Bus P-S3991 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with α-actinin antibody (secondary antibody: FITC-conjugated IgG). Bars, 5 µm. Right bar graph shows proportion of ARC exhibiting clear N2Bus P-S3991 striations or no such striated pattern, for each group. Numbers above columns indicate total number of cells included in the analysis

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Expressing, Western Blot, Two Tailed Test, Control, Cell Culture, Immunofluorescence, Staining, Phospho-proteomics

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a , b ProQ Diamond phosphoprotein vs. Sypro Ruby total protein stain of human recombinant N2Bus phosphorylated (in presence of ATP) by ERK2 ( a ), catalytic subunit of PKA ( b ), or cGMP-activated PKG ( b ), and effect of human recombinant full-length PP5, PP5 catalytic subunit (PP5c), or enzymatic dead (ED) PP5c mutant H304A on phosphorylation. ‘Control’ is recombinant construct but no ATP/enzyme. n = 5. c 32 P-ATP autoradiography using N2Bus phosphorylated by cGMP-activated PKG and dephosphorylated by PP5 or PP5c. ‘No PK/PP’ is control in presence of 32 P-ATP, but no enzyme. n = 4. d Western blot (WB) of permeabilized human (donor) heart tissue treated with various enzymes, as follows: alkaline phosphatase (AP), cGMP-activated PKG, then PP5 or PP5c. Detection of titin phosphorylation with phospho-specific antibody to P-S4185 in human N2Bus. PVDF was coomassie-stained to reveal loading on gel. WB signals were normalized to PVDF signals. n = 4. e Site-specific phosphorylation of residues within N2Bus and PEVK titin regions by western blot in myocardial tissue from human donor (Don) and end-stage failing (Fail) hearts ( n = 10/group). Detection with phospho-specific antibodies to P-S4010, P-S4099, P-S4185, P-S11878, and P-S12022 in the human titin sequence. WB signals were normalized to PVDF signals. f Mean PP5 expression in human donor vs. end-stage failing hearts ( n = 10/group). In a – f , bar graphs show relative phosphorylation indexed to the respective phosphorylated controls. Data are mean ± s.e.m.; * p < 0.05, ** p < 0.01, and *** p < 0.001, by two-tailed Student’s t -test or Holm-Sidak method

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Staining, Recombinant, Mutagenesis, Phospho-proteomics, Control, Construct, Autoradiography, Western Blot, Sequencing, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Expression level of PP5 (left) and phospho-Raf1 S338 (right) in PP5 TG vs. WT mouse hearts by western blot. mean ± s.e.m., n = 4 hearts/group (age 5–6 months); duplicate analysis/group. b Sarcomeric localization of PP5 in PP5 WT and TG hearts by immunogold electron microscopy. Bars, 500 nm. Bar graph shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data are mean ± s.e.m., n = 5 ROIs from 2 hearts/group. c PP5 localization in cardiomyocytes from PP5 TG and WT mouse hearts by indirect immunofluorescence. PP5 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK (titin) antibody (secondary antibody: FITC-conjugated IgG). Bars, 2 µm (main) and 1 µm (insets). d PP5 overexpression specifically decreases titin phosphorylation at N2Bus in PP5 TG vs. WT hearts. Total titin phosphorylation measured by ProQ Diamond/Sypro Ruby staining (upper left), site-specific titin phosphorylation detected by western blot using antibodies to P-S3991, P-S4043, P-S4080 (all N2Bus; right panels), P-S2080 (titin Z/I junction), and P-S12742 (PEVK region). Phospho-titin signals were normalized to total titin signals detected by WB using a panel of sequence-specific antibodies (Pan). Means were indexed to those of control (WT) groups. Data are mean ± s.e.m., n = 4 hearts/group, samples analyzed in triplicate. e Localization of phospho-N2Bus P-S3991 in cardiomyocytes from PP5 TG and WT hearts by indirect immunofluorescence. Anti-N2Bus P-S3991 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK antibody (secondary antibody: FITC-conjugated IgG). Bars, 2 µm (main) and 1 µm (insets). f Sarcomeric localization of phospho-N2Bus P-S3991 in PP5 WT and TG hearts by immunogold electron microscopy. Bars, 500 nm (main) and 100 nm (insets). Bar graph shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data are mean ± s.e.m., n = 5 ROIs from 2 hearts/group. In a and d , bar graphs show relative signal changes indexed to the respective controls. In a , b , d , and f , * p < 0.05 and *** p < 0.001, by two-tailed Student’s t -test

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Expressing, Western Blot, Electron Microscopy, Immunofluorescence, Over Expression, Phospho-proteomics, Staining, Sequencing, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Representative image of permeabilized cardiomyocyte glued at the ends to a force transducer and micromotor, respectively, and experimental protocol. Bar, 20 µm. b Passive tension ( F passive ) vs. SL curves of permeabilized single PP5 TG and WT cardiomyocytes in relaxing solution, before and after treatment with recombinant PP5c. c F passive -SL curves of permeabilized single PP5 TG and WT cardiomyocytes before and after treatment with ERK2. d F passive –SL curves of permeabilized single WT cardiomyocytes before and after treatment with ERK2 and additional exposure to PP5c. Data are mean ± s.e.m., n = 5 cells/condition (2 different hearts/condition). Curves are second-order polynomial fits to the means. * p < 0.05, TG vs. WT; # p < 0.05, WT + PP5c vs. WT in b and WT + ERK2 vs. WT in c and d ; † p < 0.05, TG + ERK2 vs. TG in c and WT + ERK2 + PP5c vs. WT + ERK2 in d ; all by two-tailed Student’s t -test

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Interactions of PP5 or PP5c by GST-pulldown assay. The PP5-GST assay is a negative control. b Interactions of N2Bus by GST-pulldown assay. c Impact of Hsp90 on PP5-N2Bus binding in GST-pulldown ‘competition’ assays. ‘PP5→Hsp90’: N2Bus immobilized on GSH-beads, incubated with PP5, then washed, and Hsp90 added thereafter. ‘Hsp90→PP5’: N2Bus immobilized on GSH-beads, incubated with Hsp90, then washed, and PP5 added thereafter. Summary data in bar graph are relative values indexed to the ‘PP5→Hsp90’ condition; mean ± s.e.m., n = 5 experiments/condition; * p < 0.05, by two-tailed Student’s t -test. d Localization of Hsp90 in cardiomyocytes from PP5 TG and WT hearts by indirect immunofluorescence. Anti-Hsp90 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK (titin) antibody (secondary antibody: FITC-conjugated IgG). Bars, 5 µm (main) and 1 µm (inset)

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: GST Pulldown Assay, Glutathione S-Transferase Assay, Negative Control, Binding Assay, Incubation, Two Tailed Test, Immunofluorescence

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: a Total titin phosphorylation measured by ProQ Diamond/Sypro Ruby staining (left) and site-specific titin phosphorylation detected by western blot using antibodies to P-S3991 (N2Bus; middle) or P-S2080 (Z/I junction; right). Site-specific titin phosphorylation levels were normalized to total titin levels detected by WB using sequence-specific antibodies (Pan). Means were indexed to those of control (WT) groups. Data are mean ± s.e.m., n = 3 hearts/group, samples analyzed in triplicate; * p < 0.05, by two-tailed Student’s t -test. b , c , d Localization of PP5 ( b ), phospho-N2Bus P-S3991 ( c ), and phospho-Z/I-junction P-S2080 ( d ) in cardiomyocytes from FHL-1 WT and KO hearts by indirect immunofluorescence. Anti-PP5, anti-N2Bus P-S3991 or anti-Z/I-junction P-S2080 (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK antibody (secondary antibody: FITC-conjugated IgG). Bars, 5 µm (main) and 1 µm (insets). e , f Sarcomeric localization of phospho-N2Bus P-S3991 ( e ) and phospho-Z/I-junction P-S2080 ( f ) in FHL-1 WT and KO hearts by immunogold electron microscopy. Bars, 500 nm (main) and 100 nm (insets). Bar graph in e and f shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data in e and f are mean ± s.e.m., n = 5 ROIs from two hearts/group. In a and e , * p < 0.05 and *** p < 0.001, by two-tailed Student’s t -test

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Phospho-proteomics, Staining, Western Blot, Sequencing, Control, Two Tailed Test, Immunofluorescence, Electron Microscopy

Journal: Nature Communications

Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

doi: 10.1038/s41467-017-02483-3

Figure Lengend Snippet: Under basal conditions, PP5 activity towards titin N2Bus and MAPK/ERK family member Raf1 is low and the relatively high distensibility of N2Bus results in relatively low titin-based passive tension (left side). The strain-dependent mechanosensor connecting MAPKs to N2Bus via FHL-1 functions normally, as downstream signaling from Raf1 to ERK2 is enabled. When PP5 expression is increased (as in failing hearts) and PP5 becomes activated through interaction with Hsp90, Ca 2+ /S100 protein, arachidonic acid (aa), or long chain fatty acid-CoA esters (LCACE), the phosphatase translocates to the I-band mechanosensor at N2Bus (right side). Thus, N2Bus (previously phosphorylated by ERK2, PKA, PKG, or CaMKII) is dephosphorylated, which reduces its distensibility and increases titin-based passive tension; the mechanosensor is now less sensitive. Raf-1 is also dephosphorylated and signaling to ERK2 is disabled, such that the mechanosensor function is additionally compromised. The process is embedded in signaling pathways activated via G-protein coupled receptor (GPCR) and Ras, and it can be reversed when PP5 is deactivated. (Molecules that have a color code were studied here, those with no color/white background were inferred from the literature)

Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr 202 /Tyr 204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

Techniques: Activity Assay, Expressing, Protein-Protein interactions

Journal: Oncotarget

Article Title: Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer.

doi: 10.18632/oncotarget.3268

Figure Lengend Snippet: Figure 3: CUDC-101 inhibits ATC cell proliferation, and induces cell cycle arrest and apoptosis. (A) Basal expression of HDAC1, HDAC2 and EGFR in ATC cell lines. (B) Cell proliferation assay. Error bars are mean ± SD. (C) Cell cycle analysis after 24 hours of treatment. (D) The Caspase-Glo 3/7 assay after 48 hours of treatment with CUDC-101. *p < 0.05, **p < 0.01, ***p < 0.001. NS, no significant difference.

Article Snippet: Phospho-kinase and apoptosis protein arrays Human phospho-kinase (Catalog # ARY003B) array (detecting site-specific phosphorylation of 43 kinases and 2 related total proteins, and apoptosis array (Catalog # ARY009 detecting the expression level of 35 apoptosisrelated proteins) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Proliferation Assay, Cell Cycle Assay, Caspase-Glo Assay

Journal: Oncotarget

Article Title: Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer.

doi: 10.18632/oncotarget.3268

Figure Lengend Snippet: Figure 5: Protein targets of CUDC-101 in ATC cells. (A) CUDC-101 inhibits HDACs and MAPK in ATC cells. (B) Targets of CUDC-101 identified by phospho-kinase array. Phospho-kinase arrays were performed using cell lysates treated with and without CUDC-101. Shown are those proteins that were altered with CUDC-101 treatment in both ATC cell lines with > 1.5-fold difference. (C) Differentially expressed apoptotic proteins with and without CUDC-101 treatment. Apoptosis arrays were performed. Shown are those proteins that were altered with CUDC-101 treatment in both ATC cell lines with > 1.5-fold difference. For A–C, ATC cells were treated with the vehicle or CUDC-101 at 1.1 μM for 24 hours. (D) CUDC-101 reduces the expression of survivin, XIAP, and β-catenin. Cells were treated with the vehicle or CUDC-101 for 24 hours.

Article Snippet: Phospho-kinase and apoptosis protein arrays Human phospho-kinase (Catalog # ARY003B) array (detecting site-specific phosphorylation of 43 kinases and 2 related total proteins, and apoptosis array (Catalog # ARY009 detecting the expression level of 35 apoptosisrelated proteins) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing