Col1a2

Manufactured by Santa Cruz Biotechnology

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COL1A2 is a collagen type I alpha 2 chain protein that is a structural component of type I collagen. Type I collagen is the most abundant protein in the human body and is a major component of connective tissues such as skin, bone, and tendon.

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7 protocols using col1a2

Quantitative Telomere-Damage Analysis in Lung Cells

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After deparaffinization and rehydration, tissues underwent antigen retrieval in 10 mM sodium citrate buffer and permeabilization was performed in PBS 0.5% Triton X-100 for 3 h. Next, tissues were washed 3 × 5 min in PBS 1X, fixed in 4% formaldehyde for 5 min, washed 3 × 5 min in PBS and dehydrated in a 70%–90%–100% ethanol series (5 min each). Then, the immuno-telomere-Q-FISH with COL1A2 (Clone E-6 1:400, Santa Cruz Biotechnology), SCGB1A1/CC10 (Clone E-11 1:100, Santa Cruz Biotechnology) and p63 (Clone 4A4, Roche) antibodies was performed and analyzed as previously described^{50 (link),71 (link)}. Following the same protocol, an immuno-telomere-Q-FISH with the DNA damage marker 53BP1 (1:500, Novus Biologicals, Centennial, CO) was performed to identify telomeric induced foci (TIF) in COL1A2 (Clone E-6 1:400, Santa Cruz Biotechnology), SCGB1A1 (Clone E-11 AF488 1:100, Santa Cruz Biotechnology) and p63 (Clone 4A4, Roche, Basel, Switzerland) positive cells as previously described^{71 (link),73 (link)}.

Piñeiro-Hermida S., Martínez P., Bosso G., Flores J.M., Saraswati S., Connor J., Lemaire R, & Blasco M.A. (2022). Consequences of telomere dysfunction in fibroblasts, club and basal cells for lung fibrosis development. Nature Communications, 13, 5656.

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Protein Detection in Cell Lysates

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Samples were lysed in lysis buffer (50 mM Tris (pH 7.4), 150 mM KCl, 1 mM EDTA, 1% NP-40, 5 mM NAM, 1 mM sodium butyrate, protease and phosphatase inhibitors). Proteins were separated by SDS–PAGE and transferred onto nitrocellulose or polyvinylidene difluoride membranes. Blocking (30 min) and antibody incubations (overnight) were performed in 5% BSA in TBST. YAP1 (Santa Cruz sc101199, 1:1000), phospho-YAP1^Y357 (Abcam ab62751, 1:1000), ACTIN (Santa Cruz sc47778, 1:1000), CXCL10 (RD system AF-466-NA, 1:1000), CCL2 (Novus Biologicals NBP2-22115, 1:1000), COL1A2 (Santa Cruz, 1:1000).

Sorrentino G., Rezakhani S., Yildiz E., Nuciforo S., Heim M.H., Lutolf M.P, & Schoonjans K. (2020). Mechano-modulatory synthetic niches for liver organoid derivation. Nature Communications, 11, 3416.

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Protein Expression Analysis via Western Blot

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Whole cell lysate (40–50 μg proteins) from each sample was mixed with sample loading buffer and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrotransferred onto a polyvinylidene fluoride membrane, and blocked with 1x Tris-buffered saline-Tween 20 (TBST) containing 5% fat-free milk for 1 h at room temperature and then incubated overnight at 4°C in TBST containing 3% fat-free milk with primary antibodies (1:250 dilution). The membrane was treated with corresponding secondary anti-mouse or anti-chicken horseradish peroxidase-conjugated antibodies (1:5000 dilutions). Protein bands were developed using a SuperSignal West Femto Chemiluminescent kit and visualized using an Alpha Innotech detection system from Proteinsimple (Santa Clara, CA, USA). The intensity of protein bands was quantified by Alphaview software. The primary antibodies; δEF1 (catalog # sc-10573), pSmad2/3 (Ser423/425; catalog # sc-11769), H1 (catalog # sc-10806), α-SMA (catalog # A-7607), COL1A2 (catalog # sc-8788), and β-actin (catalog # A5316) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Primary antibody for NPRA was produced as previously described [16 (link), 65 ].

Sen A., Kumar P., Garg R., Lindsey S.H., Katakam P.V., Bloodworth M, & Pandey K.N. (2016). Transforming Growth Factor-β1 Antagonizes the Transcription, Expression, and Vascular Signaling of Guanylyl Cyclase/Natriuretic Peptide Receptor A: Role of δEF1. The FEBS journal, 283(9), 1767-1781.

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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 of Cell Lysates

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Cell lysates were prepared with PRO-PREP™ Protein Extraction Solution (iNtRON Bio-technology, Gyeonggi do, Korea). Total proteins were separated with a NuPAGE electrophoresis system (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed using primary antibody against p16INK4A, p21Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA), MMP1, Col1A2, and β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Scanning densitometric values of bands were analyzed using the ImageJ, software version 1.52a (National Institutes of Health, Bethesda, MD, USA).

Kim Y., Ji H., Cho E., Park N.H., Hwang K., Park W., Lee K.S., Park D, & Jung E. (2021). nc886, a Non-Coding RNA, Is a New Biomarker and Epigenetic Mediator of Cellular Senescence in Fibroblasts. International Journal of Molecular Sciences, 22(24), 13673.

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Protein Expression in Bone Callus

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The expression of collagen type I alpha 2 chain (COL1A2), Runx 2, OPG, and RANKL in callus was determined by western blot analysis. Total proteins were exacted by a bone tissue protein extraction kit (BB-31813, BestBio Biotechnology Co., Ltd., Shanghai, China), and protein concentration was evaluated using a bicinchoninic acid (BCA) protein quantification kit (Beyotime Institute of Biotechnology, Shanghai, China), in line with the instructions provided. Equal amounts of protein (40 μg) were separated by electrophoresis and electroblotted onto a 0.45 μm membrane. COL1A2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Runx 2 (Abcam, Cambridge, UK), OPG (Abcam), and RANKL (Abcam) were detected with specific primary and secondary antibodies. The dilutions of the primary antibodies were as follows: COL1A2, 1: 500; Runx 2, 1: 500; OPG, 1: 1000; RANKL, 1: 500. The dilutions of the secondary antibodies were as follows: COL1A2, 1: 10 000; Runx 2, 1: 10 000; OPG, 1: 10 000; RANKL, 1: 10 000. Western blot analysis was performed according to standard procedures [10 (link)]. The optical density of the western blot bands was then determined with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Zhang Y., Han B., Wei Y., Jing J, & Li J. (2020). Icariin Promotes Fracture Healing in Ovariectomized Rats. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e924554-1-e924554-8.

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Molecular Profiling of ECM Regulators

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The reagents, antibodies, and materials used in this study were purchased as follows: Life Technologies (Austin, TX, United States)—TRIzol^TM LS Reagent (10296028); Zymo Research (Irvine, CA, United States)—Direct-zolTM RNA MiniPrep (R2050S); primary antibodies: Novus Biologicals LLC (Centennial, CO, United States)—anti-MKL2 (NBP1-46209) and anti-SREBP2 (NBP1-54446); Santa Cruz Biotechnology, (Dallas, TX, USA)—anti-HMGCS1 (sc-166763), anti-PAI-1 (sc-5297), anti-COL1A2 (sc-393573), and anti-GAPDH (sc-25778); rabbit anti-laminin HB-4 (anti-laminin-1 sera recognizing the total laminin including α1, β1, and γ1) (LeMosy et al., 1996 (link)) (gift from Dr. Harold Erickson, Duke University). Secondary antibodies conjugated with horseradish peroxidase (Jackson Immuno Research, West Grove, USA)—Donkey anti-mouse IgG (715-035-150) and anti-rabbit IgG (715-035-144). Cell Signaling Technology (Danvers, MA, United States) RIPA Buffer (9806S). GE Healthcare (Chicago, IL, United States)—nitrocellulose membrane (10600003).

Soundararajan A., Wang T., Sundararajan R., Wijeratne A., Mosley A., Harvey F.C., Bhattacharya S, & Pattabiraman P.P. (2022). Multiomics analysis reveals the mechanical stress-dependent changes in trabecular meshwork cytoskeletal-extracellular matrix interactions. Frontiers in Cell and Developmental Biology, 10, 874828.

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