camp response element binding protein creb1  (Proteintech)

Journal: International Journal of Nanomedicine

Article Title: Tadalafil Enhances the Therapeutic Efficacy of Mesenchymal Stem Cells-Derived Exosomes in Pulmonary Hypertension by Upregulating miR-29a-3p

doi: 10.2147/IJN.S493047

Figure Lengend Snippet: MiR-29a-3p regulates inflammation through ENPP2; ( A ) The expression values for miR-29a-3p in MSCs treated by PBS, TAD, TAD (antagomir), or agomir tested by RT-qPCR. *P < 0.05, ***P < 0.001 vs MSC; # P < 0.05 vs MSC TAD ; ( B ) The expression values for miR-29a-3p in MSC-Exo treated by PBS, TAD, TAD(antagomir), or agomir tested by RT-qPCR. **P < 0.01, ***P < 0.001 vs MSC-Exo; ## P < 0.01 vs MSC TAD- Exo; ( C ) The expression values for miR-29a-3p in RAW264.7 treated by different MSC-Exo. ***P < 0.001 vs LPS; ## P < 0.01 vs LPS+MSC-Exo; ^ P < 0.05 vs LPS+MSC TAD -Exo; ( D – F ) ELISA for the expression of pro-inflammatory factors IL-6, TNFα, and IL-1β. *P < 0.05, **P < 0.01 vs LPS+MSC TAD -Exo; ( G ) The protein expression of ENPP2 after treatment with different groups of exosomes. *P < 0.05, ***P < 0.001 vs LPS; # P < 0.05 vs LPS+MSC-Exo; ^ P < 0.05 vs LPS+MSC TAD -Exo; ( H ) One region in the enpp2 3’-UTR was predicted to bind with miR-29a-3p. ***P < 0.001 vs.NC (agomir)+ 3’-UTR-wt; ### P < 0.001 vs miR-29a-3p+3’-UTR-wt; ( I ) Proinflammatory factor protein expression in different treatments. ( A – I ) n = 3. ***P < 0.001 vs LPS+ MSC TAD -Exo; ### P < 0.001 vs LPS+MSC (agomir) -Exo.

Article Snippet: China’s Genechem Co., Ltd. provided the small interfering RNAs (siRNAs) for cAMP-responsive element binding protein 1 (CREB1), CEBPα, and Enpp2.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: PKCε Signalling Activates ERK1/2, and Regulates Aggrecan, ADAMTS5, and miR377 Gene Expression in Human Nucleus Pulposus Cells

doi: 10.1371/journal.pone.0082045

Figure Lengend Snippet: Western blot analyses of NP cell lysates (25 µg of protein per lane) treated with vehicle (time 0) or with the PKCε-selective activator peptide ψεRACK (1μM) for 8, 18, or 40 hours show ( A ) ψεRACK-dependent increases in phosphorylation of PKCε (P-PKCε, left upper panel), and of the PKC-specific substrate MARCKS (P-MARCKS, right upper panel) (lower panels show PKCε and MARCKS abundance, respectively); ( B ) time course of PKCε-induced ERK1/2 phosphorylation, which establishes a moderate and sustained ERK activation up to 40 hours (left upper panel) (lower panel shows ERK1/2 protein abundance), and of PKCε activation-dependent increases in expression of the transcription factor CREB1 at 8 hours (right panel); ( C ) significant increases in expression of transcription factors Fos and ATF also at 8 hours after exposure to ψεRACK; equal loading for CREB1, Fos, and ATF was additionally monitored with immunodetection of p120GAP (right panel) on the upper part of the nitrocellulose membranes; ( D ) that ψεRACK-dependent ERK, CREB1, ATF and Fos activation was abolished by U0126 and PD98059, both inhibitors of MEK1/2, but not SB203580 (all inhibitors were added 20 min prior to treatments with ψεRACK). Similar results were obtained in five different NP cell lines; examples shown are from NP13 P8-P12 (A-C) and from NP11 P9-P13 (D).

Article Snippet: We used the following antibodies (and dilutions): polyclonal antibodies to tropomyocin (1:400) from Sigma, to phosphorylated species of PKCε (1:1,000) and myristoylated alanine-rich C-kinase substrate (MARCKS) (1:1,000), to PKCε (1:1,000), ERK1/2 (1:2,000), and to activator protein 1 (AP-1) (1:1,000) from Santa Cruz, to cAMP responsive element binding protein 1(CREB1) (1:1,000) from Millipore; mouse monoclonal antibodies to MARCKS (1:1,000) and phosho-ERK1/2 (1:2,000) from Santa Cruz, to p120GAP (1:2,000) from Upstate Biotechnology, to neo-epitope ARG on aggrecan, clone BC-3 (1:1,000 for Western blotting and 1:200 for immunocytochemistry) from ABCAM, and to β-tubulin (1:500) from Sigma; and secondary antibodies conjugated to horseradish peroxidase or fluorochromes from Santa Cruz.

Techniques: Western Blot, Activation Assay, Expressing, Immunodetection

Journal: PLoS ONE

Article Title: PKCε Signalling Activates ERK1/2, and Regulates Aggrecan, ADAMTS5, and miR377 Gene Expression in Human Nucleus Pulposus Cells

doi: 10.1371/journal.pone.0082045

Figure Lengend Snippet: Genomic pairwise sequence alignments which correspond to the region that spans 1500bp upstream of the human transcription start site of ACAN and ADAMTS5, or the intergenic region (1375bp) between hsa-mir-377 and its 5’ adjacent hsa-mir-496 were used to identify potential AP1 (red) and CREB1 (green) transcription binding sites. Comparative analysis with four other animal species, namely Oryctolagus cuniculus-rabbit, Rattus norvegicus-rat, Canis familiaris-dog, and Bos taurus-cow that are commonly used for IVD studies, revealed that the ACAN and ADAMTS5 promoters in Homo sapiens are less responsive to AP-1 and CREB activation than the bovine ones, in contrast to the hsa-mir-377 promoter which is enriched in these sites and thus expected to yield higher stoichiometry responses (“--” indicate non available alignments).

Article Snippet: We used the following antibodies (and dilutions): polyclonal antibodies to tropomyocin (1:400) from Sigma, to phosphorylated species of PKCε (1:1,000) and myristoylated alanine-rich C-kinase substrate (MARCKS) (1:1,000), to PKCε (1:1,000), ERK1/2 (1:2,000), and to activator protein 1 (AP-1) (1:1,000) from Santa Cruz, to cAMP responsive element binding protein 1(CREB1) (1:1,000) from Millipore; mouse monoclonal antibodies to MARCKS (1:1,000) and phosho-ERK1/2 (1:2,000) from Santa Cruz, to p120GAP (1:2,000) from Upstate Biotechnology, to neo-epitope ARG on aggrecan, clone BC-3 (1:1,000 for Western blotting and 1:200 for immunocytochemistry) from ABCAM, and to β-tubulin (1:500) from Sigma; and secondary antibodies conjugated to horseradish peroxidase or fluorochromes from Santa Cruz.

Techniques: Sequencing, Binding Assay, Activation Assay