Journal: Developmental Biology

Article Title: Developmental regulation of MURF ubiquitin ligases and autophagy proteins nbr1, p62/SQSTM1 and LC3 during cardiac myofibril assembly and turnover

doi: 10.1016/j.ydbio.2010.12.024

Figure Lengend Snippet: Developmental expression of MURF-family members and related UPS/autophagy proteins in the heart. A. Western blot analysis of mouse heart lysates from embryonic (E) to postnatal (P) stages. The MURF2 isoforms are marked as p50A and p60A (here: detected with goat anti-MURF2) Full-length SRF (FL) and the repressive isoform SRF-Δ5. B. Plot of the densitometric analysis on Western blots normalised for GAPDH levels. C. RT-PCR on cDNA isolated from mouse hearts with the full-length MURF2 and MURF1 primers shows developmentally regulated levels and splice variant expression for MURF2. D. Plot of RT-PCR data obtained from 3 independent sets of experiments show the late-onset of MURF1 expression; error bars represent standard deviation. Data are normalised for GAPDH expression E. Western blots showing the differential expression of MURF2 p50A and p60A isoforms between the left ventricle (LV) and left atrium (LA) between postnatal day P9 and adult stages. E: embryonic day; P: postnatal day; Wk: week.

Article Snippet: Additionally, rabbit anti-p62/Sequestosome1 (Abcam ab56416, 1:500), rabbit anti-LC3 (Cell Signalling #2775, 1:500), mouse monoclonal anti-nbr1 (Abcam ab55474, 1:500), rabbit anti-SRF (Santa-Cruz SC13029, 1:500), goat anti-MURF3 (Santa-Cruz SC50252, 1:500), rabbit anti-GAPDH (Abcam ab9485, 1:1000) and rabbit anti-actin (Sigma A2066, 1:1500) antibodies were used.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Variant Assay, Standard Deviation

Journal: Biosensors

Article Title: Central [CNS] and Peripheral [Gastric Tissue] Selective Monitoring of Somatostatin (SRIF) with Micro-Sensor and Voltammetry in Rats: Influence of Growth Factors (GH, EGF)

doi: 10.3390/bios7040053

Figure Lengend Snippet: Typical DPVoltammograms monitored in the striatum of anesthetized rats following local injection of SRIF (top, n = 1), SRIF antisera (S.A.), i.e., antibodies for SRIF (rabbit polyclonal, IgG antiSRIF AB5494); Millipore (MERCK) (middle, n = 1); or control antisera (C.A.), i.e., non specific antibodies in the striatum of a single animal (bottom, n = 1). See for data obtained in groups of rats treated as above (n = 7 each treatment), as well as with NaCl 0.9% (control group, n = 9), Bacitracin (n = 5), GH (n = 5) and Cysteamine (n = 5).

Article Snippet: - SRIF antisera, i.e., antibodies for SRIF (rabbit polyclonal, IgG antiSRIF AB5494); Millipore (MERCK), but not control antisera, i.e., non-specific antibodies, as described by Funato, et al. [ ], resulted in a rapid decrease until disappearance of striatal Peak 5.

Techniques: Injection, Control

Journal: Biosensors

Article Title: Central [CNS] and Peripheral [Gastric Tissue] Selective Monitoring of Somatostatin (SRIF) with Micro-Sensor and Voltammetry in Rats: Influence of Growth Factors (GH, EGF)

doi: 10.3390/bios7040053

Figure Lengend Snippet: ( A ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with aspecific IgG antisera (asp IgG). Note that the oxidation signal monitored at approximately .8 V (i.e., 800 mV) and measuring approximately 60 nanoAmperes (nA) superimposes the signal detected in the gastric tissue incubated with vehicle. Right, data obtained in antral preparation from 5 rats, incubated with vehicle or aspecific IgG antisera [asp IgG]; ( B ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with specific IgG antisera (SRIF IgG); note that the peak monitored at +800 mV (denoted as peak 3 in this figure) and having a size of approximately 60 nA is no longer detected following incubation with specific IgG antiSRIF [peaks 1 and 2 are detected at lower oxidation potential, i.e., +200 or +400 mV, respectively, and are not affected by SRIF IgG]. Right, data obtained in antral preparation from 5 rats, incubated with vehicle or specific IgG antisera; ( C ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with cysteamine; note that the peak monitored at +800 mV is greatly decreased. Right, data obtained in antral preparation from 5 rats, incubated with vehicle or cysteamine; ( D ) Left: DPV-µCFE scans obtained in ( a ) vehicle (PBS); ( b ) in the gastric tissue of a single animal incubated with vehicle; ( c ) in the gastric tissue of a single animal incubated with EGF: note that the peak monitored at +800 mV is significantly increased, i.e., from approximately 60 nA to approximately 85 nA.

Article Snippet: - SRIF antisera, i.e., antibodies for SRIF (rabbit polyclonal, IgG antiSRIF AB5494); Millipore (MERCK), but not control antisera, i.e., non-specific antibodies, as described by Funato, et al. [ ], resulted in a rapid decrease until disappearance of striatal Peak 5.

Techniques: Incubation

Journal: Molecular and Cellular Biology

Article Title: Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

doi: 10.1128/mcb.24.16.7298-7311.2004

Figure Lengend Snippet: FIG. 1. SRF protein levels do not change with cellular age. (A) Western blots used equal amounts of protein from young (Y, 36 mean population doublings) and old (O, 82 mean population doublings) Hs68 fibroblasts. Total (Tot) and nuclear (Nuc) extracts and a polyclonal antibody generated against full-length SRF were used. The arrow identifies a 67-kDa band characteristic of native SRF. (B) Young and senescent primary human diploid fibroblasts were fixed and stained with rabbit anti-SRF followed by goat anti-rabbit antibody–Texas Red and counterstained with DAPI to visualize DNA.

Article Snippet: Incubation with 20 ng of PKC polyclonal antibody (Santa Cruz sc-937) or 0.5 l of SRF rabbit antiserum was carried out with 500 g of total cellular protein for 4 h at 4°C.

Techniques: Western Blot, Generated, Staining

Journal: Molecular and Cellular Biology

Article Title: Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

doi: 10.1128/mcb.24.16.7298-7311.2004

Figure Lengend Snippet: FIG. 2. Kinase activity in senescent-cell nuclear extracts inhibits SRF DNA binding activity. (A) EMSAs with 32P-labeled SRE oligonucleotide and SRF(His)6 previously incubated with nuclear extracts from young (Y) or old (O) nuclear extracts are shown in lanes 1 and 2. Parallel kinase reactions were incubated with mutant (mut) SRE (lanes 3 and 4), competed with a 100-fold excess of unlabeled wild-type (WT) SRE (lanes 5 and 6), or incubated with a 100-fold excess of unlabeled mutant SRE (lanes 7 and 8). Preincubation with polyclonal SRF antibody (SRF, lanes 9 and 10) supershifted or eliminated the SRF complex. Parallel reactions without SRF(His)6 did not form complexes. (B) SRF(His)6 was used in kinase reactions with equal amounts of young (Y), senescent (O), or a combination of young- and old-cell nuclear extracts (Y/O) and used in SRE EMSAs (lanes 2 to 4). A control (C) kinase reaction with SRF but without nuclear extract is shown in lane 1. Parallel reactions were also done in the presence of the phosphatase inhibitors sodium fluoride (NaF) and sodium vanadate (Na-Van) (lanes 5 to 7). The proportional addition of senescent (Old%) nuclear extracts to young nuclear extracts (Young%) were also used in SRE EMSAs (lanes 8 to 12). (C) Reactions with SRF(His)6 incubated with kinases supplied from equal amounts of young (Y), senescent (O), or a combinations of young and old nuclear extracts (Y/O) with 10 M ATP were used in SRE EMSAs (lanes 2 to 4). A control (C) reaction with SRF but without nuclear extract is shown in lane 1. Parallel reactions were also carried out in the absence of ATP used in EMSAs (lanes 5 to 8). (D) Data were obtained from SRE EMSAs utilizing SRF kinase reactions in the presence of SRF(His)6 as the substrate and kinases supplied from equal amounts of young (Y) and senescent (O) nuclear extracts. Various amounts of the PKC inhibitors bisinodolylmaleimide II (Bis II; 25, 50, and 100 nM), chelerythrine chloride (CH-Cl; 1.25, 2.5 and 5 M), rottlerin (Rot; 5, 10, and 20 M), or dimethyl sulfoxide (DMSO, 1%) vehicle were used in each reaction. Control reactions with SRF but in the absence of nuclear extracts were also followed by EMSA and used to normalize experiments. Histograms show data from scanning densitometry of three independent EMSAs with the average ratio of young and old intensities relative to the control reaction under each drug concentration, with standard deviations shown by error bars.

Article Snippet: Incubation with 20 ng of PKC polyclonal antibody (Santa Cruz sc-937) or 0.5 l of SRF rabbit antiserum was carried out with 500 g of total cellular protein for 4 h at 4°C.

Techniques: Activity Assay, Binding Assay, Labeling, Incubation, Mutagenesis, Control, Concentration Assay

Journal: Molecular and Cellular Biology

Article Title: Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

doi: 10.1128/mcb.24.16.7298-7311.2004

Figure Lengend Snippet: FIG. 3. Specific PKC kinase inhibitors and activators modulate SRF DNA binding activity. (A) In vitro kinase reactions with SRF(His)6 and kinases supplied from equal amounts of young (Y), senescent (O), or a combination of young- and old-cell nuclear extracts (Y/O) were performed in the presence of [-32P]ATP. Control (C) reactions with SRF but without nuclear extract were also incubated in the presence of [-32P]ATP (lanes 1, 5, and 9). Parallel sets of reactions were carried out in the presence of either 20 M rottlerin (lanes 5 to 8) or 50 nM bistratene A (lanes 9 to 12), which inhibit and activate PKC, respectively (B). In vitro kinase reactions performed in parallel without radiolabel were used with labeled SRE EMSAs (lanes 13 to 24).

Article Snippet: Incubation with 20 ng of PKC polyclonal antibody (Santa Cruz sc-937) or 0.5 l of SRF rabbit antiserum was carried out with 500 g of total cellular protein for 4 h at 4°C.

Techniques: Binding Assay, Activity Assay, In Vitro, Control, Incubation, Labeling

Journal: Molecular and Cellular Biology

Article Title: Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

doi: 10.1128/mcb.24.16.7298-7311.2004

Figure Lengend Snippet: FIG. 5. PKC activity is elevated during senescence (A) Phospho- Thr-505 Western blot of total lysates of young (Y) and old (O) cells harvested after serum starvation for 48 h or when stimulated for 0.5 h with 100 nM phorbol myristate acetate. (B) The samples used in panel A were used in a PKC Western blot (sc-937) as a loading control for the phosphorylation-specific Western blot. (C) Lysates from young and old fibroblasts were precipitated with anti-PKC antibody (sc-937), nonspecific rabbit immunoglobulin G, or beads. Kinase reactions were performed with aliquots of the immunoprecipitations described in panel B, [-32P]ATP, and SRF(His)6. (D) Precipitated PKC was vi- sualized by a Western blot with polyclonal goat anti-PKC (lanes 1 to 6). (E) Histogram of data obtained from scanning densitometry of three independent immunoprecipitation kinase reactions as described for panel C. Error bars indicate standard deviations.

Article Snippet: Incubation with 20 ng of PKC polyclonal antibody (Santa Cruz sc-937) or 0.5 l of SRF rabbit antiserum was carried out with 500 g of total cellular protein for 4 h at 4°C.

Techniques: Activity Assay, Western Blot, Control, Phospho-proteomics, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

doi: 10.1128/mcb.24.16.7298-7311.2004

Figure Lengend Snippet: FIG. 6. Recombinant PKC inhibits SRF DNA binding activity. (A) A preparative digest of PKC was performed by incubation for 3 h at 37°C with recombinant caspase 3. A PKC Western blot shows the liberation of the PKC catalytic fragment (PKC-CF). These preparative fractions were used in subsequent phosphorylation analyses with PKC. (C) Phosphorylation-specific Western blot and SRE EMSA of in vitro kinase assays with activated PKC (lanes 2 to 4), caspase-cleaved PKC (lanes 5 to 7), and recombinant casein kinase II (lanes 8 to 10). The kinases were used to phosphorylate SRF(His)6 in a time course of 45 to 180 min at 37°C. The control (lane 1) used SRF(His)6 alone under the same conditions without any kinase. Reaction products were used in SRE EMSAs (B) or in a Western blot with a phosphoserine/threonine-phenylalanine (Phe 1) antibody (C).

Article Snippet: Incubation with 20 ng of PKC polyclonal antibody (Santa Cruz sc-937) or 0.5 l of SRF rabbit antiserum was carried out with 500 g of total cellular protein for 4 h at 4°C.

Techniques: Recombinant, Binding Assay, Activity Assay, Incubation, Western Blot, Phospho-proteomics, In Vitro, Control

Journal: Molecular and Cellular Biology

Article Title: Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

doi: 10.1128/mcb.24.16.7298-7311.2004

Figure Lengend Snippet: FIG. 7. PKC phosphorylates both native and recombinant SRF on T160, and mutation of this site blocks SRF inactivation. (A) The mutant form of SRF, A160, and wild-type SRF were subjected to PKC and casein kinase II treatment for 90 min. Kinase reactions with [-32P]ATP, SRF (wild type or A160), and PKC or casein kinase II were performed, and the results are shown in the top panel. Parallel reactions without radiolabel were analyzed by Western blotting with the Phe 1 or Arg 3 phosphorylation-specific antibodies and are shown in the bottom panels. (B) Parallel unlabeled reactions were also used in SRF-SRE EMSAs. Control (C) reactions in the EMSA used SRF T160 or A160 but were not treated with kinase. (C) Native SRF phospho-analysis was performed with young- and senescent-cell extracts treated with dimethyl sulfoxide (DMSO), rottlerin (Rot), or bistratene A (BisA) before harvesting. An immunoprecipitation with the SRF polyclonal was followed by resolution by SDS–10% PAGE and transfer. Western blots of native SRF used anti-SRF (SRF), phospho-S/T Phe 1 (anti-Phe 1), or phospho-S/T Arg 3 (anti-Arg-3) antibodies. (D) Small peptides of SRF which are generated by Glu-C digestion and contain the consensus sequence for the anti-phospho-Phe 1 antibody (S/T-F, boxed) or anti-phospho-Arg 3 antibody (RxxS/T, bold). (E) Peptide analysis was carried out by immunoprecipitating SRF from young (Y) and senescent (O) cell extracts after vehicle, bistratene A (BisA), or rottlerin (Rot) treatment, silver staining, isolation from gels, and digestion with Glu-C. The resulting peptides were resolved on a 15% Tricine gel and Western blotted with phospho-S/T Phe 1 (Phe 1) or phospho-S/T Arg 3 (Arg-3) antibodies.

Article Snippet: Incubation with 20 ng of PKC polyclonal antibody (Santa Cruz sc-937) or 0.5 l of SRF rabbit antiserum was carried out with 500 g of total cellular protein for 4 h at 4°C.

Techniques: Recombinant, Mutagenesis, Western Blot, Phospho-proteomics, Control, Immunoprecipitation, Generated, Sequencing, Silver Staining, Isolation

Journal: Molecular and Cellular Biology

Article Title: Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

doi: 10.1128/mcb.24.16.7298-7311.2004

Figure Lengend Snippet: FIG. 10. Rottlerin restores immediate-early gene expression in senescent fibroblasts. (A) Young and senescent Hs68 cells were serum starved for 48 h prior to stimulation by serum for 60 to 105 min. All cells were treated for 4 h before harvest with 20 M rottlerin (lanes 11 to 20) or dimethyl sulfoxide vehicle (lanes 1 to 10). Total cellular extract was harvested and used in an Egr-1 Western blot. (B) Young (Y) and senescent (O) Hs68 fibroblasts were serum starved for 48 h prior to stimulation by serum for 60 min. Cells were treated 4 h before harvest with 20 M rottlerin, 50 nM bistratene A, or dimethyl sulfoxide vehicle. Nuclear extracts from these cells were harvested and used in an SRF-SRE EMSA (C). RNA isolated from parallel plates of cells (described for B) and used as the substrate in RT-PCRS to detect c-fos transcript levels. Glyceralde- hhyde-3-phosphate dehydrogenase (GAPDH) served as an internal control for loading, amplification, efficiency, and RNA integrity.

Article Snippet: Incubation with 20 ng of PKC polyclonal antibody (Santa Cruz sc-937) or 0.5 l of SRF rabbit antiserum was carried out with 500 g of total cellular protein for 4 h at 4°C.

Techniques: Gene Expression, Western Blot, Isolation, Control

Journal: Cell reports

Article Title: Temporal control of PDGFRα regulates the fibroblast-to-myofibroblast transition in wound healing

doi: 10.1016/j.celrep.2022.111192

Figure Lengend Snippet:

Article Snippet: Rabbit anti-SRF , Santa Cruz , Cat# 335; RRID:AB_2255249.

Techniques: Plasmid Preparation, Recombinant, Control, Western Blot, BIA-KA, SYBR Green Assay, Cell Culture, Software