Col3a1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

COL3A1 is a gene that provides instructions for making a protein component of type III collagen. Collagen is a structural protein found in various tissues throughout the body, including skin, bone, and blood vessels. The COL3A1 protein is a critical part of type III collagen, which provides strength and support to these tissues.

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8 protocols using col3a1

Protein Expression Analysis in EAM Rat Myocardium

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LV myocardium from male and female EAM rats was homogenized in Laemmli buffer (253mM Tris/HCL pH 6.8, 8% SDS, 40% glycerin, 200mM DTT, 0.4% bromophenol blue) (54 (link)). Proteins were quantified with the BCA Assay (Thermo Scientific Pierce Protein Biology, Germany). Equal amounts of total proteins were separated on SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. The membranes were immunoblotted overnight with the following primary antibodies: Col3A1 (1:400, Santa Cruz, USA), ERK (1:1000, Santa Cruz, USA), p-ERK (1:2000, Santa Cruz, USA), p38 (1:500, Santa Cruz, USA) and p-p38 (1:500, Santa Cruz, USA). Equal sample loading was confirmed through an analysis of actin (1:1500, Santa Cruz, USA). Immunoreactive proteins were detected with ECL Plus (GE Healthcare, UK) and quantified with ImageLab (Bio-Rad Laboratories, USA).

Barcena M.L., Jeuthe S., Niehues M.H., Pozdniakova S., Haritonow N., Kühl A.A., Messroghli D.R, & Regitz-Zagrosek V. (2021). Sex-Specific Differences of the Inflammatory State in Experimental Autoimmune Myocarditis. Frontiers in Immunology, 12, 686384.

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Quantifying Cardiac Gene Expression

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Real‐time PCR was performed by first extracting total RNA using the TRIzol method (Invitrogen), after which relative quantification was performed using SYBR‐green detection of PCR products in real time with the ABI ViiA 7 (Applied Biosystems Inc., Foster City, CA). The following primers were used: TNNT2, MYH6, TNNC1, NPPA, RYR2, COL1A1, COL3A1, Sp1 (sc‐29487; Santa Cruz Biotechnology, Santa Cruz, CA), and Has‐miR‐590 (478168_mir; Thermo Fisher Scientific). mRNA levels were normalized by comparison to GAPDH. miRNA levels were normalized to expression of U6 snRNA.

Singh V.P., Mathison M., Patel V., Sanagasetti D., Gibson B.W., Yang J, & Rosengart T.K. (2016). MiR‐590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Toward a Cardiomyocyte‐Like Fate by Directly Repressing Specificity Protein 1. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 5(11), e003922.

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Lung Tissue Protein Extraction and Analysis

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Upper lobe of right lungs were removed and homogenized in a tissue protein extraction reagent (Thermo, USA). The lysates were centrifuged at 16000 × g at 4 °C for 15 minutes to remove insoluble protein. The protein concentrations were determined and subsequently separated using SDS-PAGE. Separated proteins were transferred to PVDF membranes (Millipore, Germany), blocked with 5% skimmed milk, and then incubated at 4 °C overnight using the following primary antibodies: α-SMA (ab5649, Abcam, USA), COL1A1 (sc-293182, Santa Cruz, USA), COL1A2 (sc-393537, Santa Cruz, USA), COL3A1 (sc-271249, Santa Cruz, USA), GAPDH (60004, Proteintech, USA). The membranes were washed three times, incubated for 1 hour using horseradish peroxidase-conjugated secondary antibodies and then visualized using an enhanced chemiluminescence detection kit (Amersham Pharmacia, Piscataway, NJ).

Yang X., Wang Y., Zhao S., Wang R, & Wang C. (2018). Long-term exposure to low-dose Haemophilus influenzae during allergic airway disease drives a steroid-resistant neutrophilic inflammation and promotes airway remodeling. Oncotarget, 9(38), 24898-24913.

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Protein Expression Analysis in Fibroblasts

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The total proteins were extracted from fibroblasts and then quantified by BCA method (BCA protein assay kit, Beyotime, China). The total proteins from each group were loading in the SDS-PAGE gel and separated by electrophoresis. Then the proteins were transferred onto a PVDF membrane. The membrane was blocked in 5 g/L skim milk for 1 h and washed in TBS. The membrane was incubated in the appropriate monoclonal antibodies at 4°C overnight. After washing in TBS-Tween (TBST), the membrane was incubated with HRP-conjugated anti-IgG secondary antibody (dilution) at 37°C for 1 h. After washing in TBST, the target proteins were visualized by using an ECL detection kit. We used antibodies to the following: GPX1(1:1000, Abcam, UK), COL1A1(1:500, Santa Cruz, USA), COL3A1(1:500, Santa Cruz, USA), Elastin(1:500, Santa Cruz, USA), MMP-2(1:500, Santa Cruz, USA), MMP-9 (1:500, Santa Cruz, USA), TIMP-2(1:500, Santa Cruz, USA), TGF-β1 (1:1000, Abcam, UK), β-actin(1:1000, Abcam, UK) and GAPDH (1:1000, Santa Cruz, USA) were used as an internal reference control.

Hong S., Hong L., Li B., Wu D., Liu C., Min J., Guo W., Hu M., Tang J, & Li Y. (2017). The role of GPX1 in the pathogenesis of female pelvic organ prolapse. PLoS ONE, 12(8), e0181896.

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Comprehensive Antibody Database for Research

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The following antibodies were used in this study: FKBP51 (ab12671, Abcam), GR (sc-393232, Santa Cruz), p-GR (#4161, Cell Signaling), IκBα (sc-1643, Santa Cruz) IL-1β (sc-7884, Santa Cruz), IL-6 (sc-130326, Santa Cruz), MKK3 (#5674, Cell Signaling), p-MKK3/6 (#9231, Cell Signaling), MMP1 (sc-21731, Santa Cruz), NF-kB (sc-109, Santa Cruz), p-NF-kB (sc-136548, Santa Cruz), TGF-β1 (sc-31609, Santa Cruz), p-Smad2/3 (sc-11769, Santa Cruz), Sirt1 (ab189494, Abcam) Smad4 (sc-7966, Santa Cruz), COL1A1 (sc-28657, Santa Cruz), COL3A1 (sc-271249, Santa Cruz), p16 (10883-1-AP, proteintech), p21 (sc-6246, Santa Cruz), p53 (1C12) (#2524, Cell Signaling), β-gal (sc-19119, Santa Cruz), MMP-1 (sc-21731, Santa Cruz), β-actin (sc-47778, Santa Cruz), and GAPDH (sc-32233, Santa Cruz). Secondary antibodies (anti-rabbit, anti-mouse, and anti-goat) were purchased from Santa Cruz Biotechnology.

Le Q.V., Wen S.Y., Chen C.J., Huang C.Y, & Kuo W.W. (2022). Reversion of glucocorticoid-induced senescence and collagen synthesis decrease by LY294002 is mediated through p38 in skin. International Journal of Biological Sciences, 18(16), 6102-6113.

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Western Blot Protein Analysis

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Antibodies to the following proteins were used for Western blotting: Collagen I (ab34710, Abcam, Cambridge, UK), Col3a1 (sc-271249, Santa Cruz, Santa Cruz, CA, USA). Alpha Tubulin (ab89984, Abcam, UK) was used as loading control.

Assayag E., Gurt I., Cohen-Kfir E., Stokar J., Zwas D.R, & Dresner-Pollak R. (2024). Cardiac Left Ventricular miRNA-26a Is Downregulated in Ovariectomized Mice, Upregulated upon 17-Beta Estradiol Replacement, and Inversely Correlated with Collagen Type 1 Gene Expression. International Journal of Molecular Sciences, 25(10), 5153.

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Signaling Pathways in Fibrosis Regulation

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Antibodies used included: SIRT2 (Abcam, Cat. No. ab211033), CTGF (Abcam, Cat. No. ab6992), FN1 (ABclonal, Wuhan, China; Cat. No. A12977), COL1A1 (Santa Cruz, Cat. No. sc-59772), TGF-β1 (Abcam, Cat. No. ab92486), β-actin (bioss, Beijing, China; Cat. No. bs-0061R), COL3A1 (Santa Cruz, Cat. No. sc-514601), E-cadherin (BD Biosciences, Cat. No. 610182), α-SMA (Santa Cruz, Cat. No. sc-84326), p-SMAD2 (Cell Signaling Technology, Cat. No. 18338), SMAD2 (Abcam, Cat. No. ab40855), acetyl Lysine (Abcam, Cat. No. ab190479), Ubiquitin (Novus, Cat. No. NB300-129), SMURF2 (ABclonal, Cat. No. A2278), CREBBP (ABclonal, Cat. No. A14237), P/CAF (ABclonal, Cat. No. A0066), Goat Anti-Rabbit IgG H&L (HRP) (Abcam, Cat. No. ab6721), and goat anti-mouse IgG H&L (HRP) (Abcam, Cat. No. ab205719).

Yang S., Yang G., Wang X., Xiang J., Kang L, & Liang Z. (2023). SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells. Cell Death & Disease, 14(9), 646.

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Protein expression analysis by Western blot

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Tissue samples and cell cultures were lysed as described elsewhere. 5 (link) Protein samples (20-50 μg) were run on SDS-PAGE and Western blot was performed. The membranes were probed with antibodies against: 1:500 COL1A1 (No. SAB1402151, Sigma-Aldrich), 1:500 COL3A1 (sc-28888, Santa Cruz Biotechnology), 1:500 COL6A1 (sc-377143, Santa Cruz Biotechnology) and β-actin or β-tubulin as loading controls (Sigma-Aldrich).

Bertorello A.M., Pires N., Igreja B., Pinho M.J., Vorkapic E., Wågsäter D., Wikström J., Behrendt M., Hamsten A., Eriksson P., Soares-da-Silva P, & Brion L. (2015). Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1). Circulation research, 116(4).

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