anti rabbit serpine e1  (Novus Biologicals)

Journal: The Journal of Biological Chemistry

Article Title: Declining activity of serum response factor in aging aorta in relation to aneurysm progression

doi: 10.1016/j.jbc.2025.108400

Figure Lengend Snippet: Confirmation of mass spectrometry data using Western blotting and immunofluorescence staining. Western blotting ( A ) confirmed reduction of proteins in the SRF regulon, along with upregulation of Serpine1, Serpine2, and Lox, and downregulation of Nedd4. Hsp90 and H3 were used as loading controls and the same amount of protein was loaded in individual lanes. Precision plus molecular weight standards were transferred to membranes together with proteins of interest, and at least one band on the standard was included on each membrane strip. Panel B shows immunofluorescence staining of Ctrl and i8-SRF-KO aorta. Panels are arranged in groups of four, showing the protein of interest in Ctrl and i8-SRF-KO (in red ) at the top , and protein of interest plus autofluorescence from elastic lamella ( green ) and the nuclear stain (DAPI, blue ) at the bottom . Reductions were localized to the media between elastic lamellae as expected, but so were increases, including increases of Myh10 and Lox. The proteomics experiment demonstrated an increased expression of the proteoglycan aggrecan (Acan). We therefore also stained the aorta using Alcian blue ( C ), showing distinct positive staining in the media of i8-SRF-KO aorta compared to Ctrl. RT-qPCR showed upregulation of the Acan transcript, while its upstream regulator Sox9 only tended to be increased, and correlation across i8-SRF-KO and Ctrl aorta was significant ( bottom panels in C ). No increase of Alcian blue staining was seen in the caudal artery ( D ). DAPI, 4',6-diamidino-2-phenylindole; RT-qPCR, reverse transcription quantitative PCR; SRF, serum response factor.

Article Snippet: The following primary antibodies were used for detection: Myh11 (Abcam, ab53219), Mylk (Abcam, ab76092), Ppp1r12a (Cell Signaling Technology, #2634), Vcl (Abcam, ab82418), Mcam (Sigma-Aldrich, SAB5600062), Lmod1 (Proteintech, 15117-1-AP), Srf (Cell Signaling Technology, #5147), Cavin1 (Abcam, ab48824), Cavin3 (Proteintech, 16250-1-AP), Actg2 (St John's Laboratory, STJ91463), Tpm1 (Cell Signaling Technology, #3910), Tagln (Abcam, ab14106), Cav1 (Cell Signaling Technology, #3267), Cav2 (BD Transduction Laboratories, 610685), Myh10 (Cell Signaling Technology, #3404), Nedd4 (R&D Systems, MAB6218-SP), Serpine2 (Proteintech, 11303-1-AP), Lox (Cell Signaling Technology, #58135), Serpine1 (Novus Biologicals, NBP1-19773), Hsp90 (BD Biosciences, 610418), Histone H3 (Cell Signaling Technology, #4499), TAZ/Wwtr1 (Cell Signaling Technology, #4883), total YAP and TAZ (Cell Signaling Technology, #8418), Gapdh (Sigma-Aldrich (Merck), MAB374), Lats1 (Cell Signaling Technology, #3477), Lats2 (Proteintech, 20276-1-AP), Slmap (Millipore Sigma, HPA002357), P-YAP (Ser-127, Cell Signaling Technology, #4911), MOB1 (Cell Signaling Technology, #13730), P-MOB (Cell Signaling Technology, #8699), P-MST1/2 (Cell Signaling Technology, #49332), MST1 (Cell Signaling Technology, #14946), and MST2 (Cell Signaling Technology, #3952).

Techniques: Mass Spectrometry, Western Blot, Immunofluorescence, Staining, Molecular Weight, Membrane, Stripping Membranes, Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Declining activity of serum response factor in aging aorta in relation to aneurysm progression

doi: 10.1016/j.jbc.2025.108400

Figure Lengend Snippet: i8-SRF-KO mice are protected from aortopathy when Ang II is administered 12 weeks after tamoxifen injection, and this associates with accumulation of YAP/TAZ. To test the hypothesis that i8-SRF-KO mice are sensitized to aortic aneurysm formation, osmotic minipumps were implanted 12 weeks after induction with tamoxifen ( A ). The experiment was terminated after an additional 4 weeks, and the aortae were prepared for whole mount imaging in relaxing physiological buffer. Four aortic lesions were observed in the Ctrl + Ang II group ( C shows all four lesions highlighted with arrows and arrowheads and the corresponding aortic regions in the KO) and none were seen in the i8-SRF-KO + Ang II group ( C , χ 2 p < 0.05). To better understand this, the imaged aortae were used for proteomics. The resulting data were integrated with our prior proteomics data. Only 13 proteins were differentially expressed in the KO versus control comparison in the presence of Ang II as compared to 729 for the same comparison in the absence of Ang II. This was due in part to 108 proteins that changed significantly and in the same manner in the Ang II versus Ctrl and in the i8-SRF-KO versus Ctrl comparisons ( D ). This is more clearly illustrated in panel E where fold-changes for these comparisons are plotted, showing that Serpine1 was among the most highly increased proteins with both interventions, and that Pdlim4 was among the most highly repressed proteins. Panel F shows protein level data for Lox, Serpine2, and Serpine1. In these cases, KO and Ang II independently increased protein levels, and only in one case (Serpine1) was an additive effect seen. Thus, depletion of Srf in the aorta mimics a molecular signature of Ang II treatment. To test if this molecular signature was governed by YAP and TAZ, we searched our proteomics data for YAP/TAZ target genes and identified several that were increased ( G ). Moreover, Western blotting ( H ) demonstrated increased levels of YAP/TAZ. For quantitative analysis, all bands, the YAP band at 70 kDa and the TAZ doublet at 50 kDa were normalized to Gapdh in the same lane, and subsequently to the mean value for the controls. Total YAP/TAZ and YAP were significantly increased ( I ). Individual symbols in scatter plots represent one animal. SRF, serum response factor; TAZ, WW domain containing transcription regulator 1; YAP, Yes-associated transcriptional regulator.

Article Snippet: The following primary antibodies were used for detection: Myh11 (Abcam, ab53219), Mylk (Abcam, ab76092), Ppp1r12a (Cell Signaling Technology, #2634), Vcl (Abcam, ab82418), Mcam (Sigma-Aldrich, SAB5600062), Lmod1 (Proteintech, 15117-1-AP), Srf (Cell Signaling Technology, #5147), Cavin1 (Abcam, ab48824), Cavin3 (Proteintech, 16250-1-AP), Actg2 (St John's Laboratory, STJ91463), Tpm1 (Cell Signaling Technology, #3910), Tagln (Abcam, ab14106), Cav1 (Cell Signaling Technology, #3267), Cav2 (BD Transduction Laboratories, 610685), Myh10 (Cell Signaling Technology, #3404), Nedd4 (R&D Systems, MAB6218-SP), Serpine2 (Proteintech, 11303-1-AP), Lox (Cell Signaling Technology, #58135), Serpine1 (Novus Biologicals, NBP1-19773), Hsp90 (BD Biosciences, 610418), Histone H3 (Cell Signaling Technology, #4499), TAZ/Wwtr1 (Cell Signaling Technology, #4883), total YAP and TAZ (Cell Signaling Technology, #8418), Gapdh (Sigma-Aldrich (Merck), MAB374), Lats1 (Cell Signaling Technology, #3477), Lats2 (Proteintech, 20276-1-AP), Slmap (Millipore Sigma, HPA002357), P-YAP (Ser-127, Cell Signaling Technology, #4911), MOB1 (Cell Signaling Technology, #13730), P-MOB (Cell Signaling Technology, #8699), P-MST1/2 (Cell Signaling Technology, #49332), MST1 (Cell Signaling Technology, #14946), and MST2 (Cell Signaling Technology, #3952).

Techniques: Injection, Imaging, Control, Comparison, Western Blot