Molecular imager chemidoc xrs imaging system

Protein was extracted from the lysed VCMs as previously described [18 (link)]. The total protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, 23225). Equal amounts of protein were separated by electrophoresis on SDS-PAGE gels (Beyotime, P0012A) and then blotted onto nitrocellulose membranes. Then, the membranes were incubated with primary antibodies overnight. The following primary antibodies were used for the western blotting: GRP78 (1:1000, Cell Signaling, Danvers, MA, USA, 3177), CHOP (1:1000, Cell Signaling, 2895), calpain (1:1000, Cell Signaling, 2539), cTnT (1:1000, Cell Signaling, 5593), caspase-3 (1:1000, Cell Signaling, 9662), Bcl-2 (1:1000, Cell Signaling, 3498), Bax (1:1000, Cell Signaling, 2772), L-type calcium channel (1:1000, Abcam, ab96713), β-tubulin (1:1000, Cell Signaling, 2148) and GAPDH (1:1000, Cell Signaling, 2118S). Secondary antibodies used were anti-rabbit IgG, HRP-linked antibody (1:5000, Cell Signaling, 7074P2) and anti-mouse IgG, HRP-linked antibody (1:5000, Cell Signaling, 7076). After the addition of the chemiluminescent reagents (Thermo Fisher Scientific, 17295), the bands were detected using a Molecular Imager ChemiDoc™ XRS+ Imaging System (Bio-Rad) and quantified using the Image Lab™ Software (Bio-Rad).

Heart tissue or NRCMs were lysed using RIPA buffer (89900, Thermo Fisher Scientific, Waltham, USA). Protein concentration was determined using a Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher Scientific). SDS-PAGE was performed with 10% gradient gel (PG112, Epizyme, China). Proteins were transferred to a PVDF membrane (#0301004001, Roche, USA). Membrane was incubated with primary antibodies at 4°C overnight after being blocked with 2% BSA for 2 hours. Primary antibodies were as follows: Nr2f2 (#6434, 1 : 1000, CST, Boston, MA, USA, Rabbit mAb), GPX4 (ab125066, 1 : 1000, Abcam, rabbit), PTGS2 (#12282, 1 : 1000, CST, Rabbit mAb), β-actin (cat no. 66009-1-Ig, 1 : 5000, Proteintech, Rosemont, IL, USA, monoclonal antibody), PGC-1α (cat no. 66369-1-Ig, 1 : 1000, Proteintech, monoclonal antibody), and GΑPDH (cat no. 60004-1-Ig, 1 : 5000, Proteintech, monoclonal antibody). Secondary antibodies were as follows: anti-rabbit IgG, HRP-linked antibody (7074P2, 1 : 5000, CST), and anti-mouse IgG, HRP-linked antibody (7076, 1 : 5000, CST). Chemiluminescent reagents (17295, Thermo Fisher Scientific) and a Molecular Imager ChemiDoc™ XRS+ Imaging System (Bio-Rad, California, USA) were used for detection. Relative protein expression was quantified by ImageJ software (ImageJ 1.8.0, Bethesda, MD, USA).

Protein lysates were prepared by incubating cells in RIPA buffer on ice for 30 min, followed by sonication using Q700 Sonicator (QSonica, Newtown, CT, USA). Proteins were resolved by SDS-PAGE and transferred to PVDF membrane using standard protocols. Following primary antibodies were used at a 1:1000 dilution (except Lamin B, which was used at 1:2000 dilution) in this study: UBF (sc-13125, Santa Cruz Biotechnology, Dallas, TX, USA); RUNX1 (4334S, Cell Signaling Technologies, Danvers, MA, USA); Cyclin B (4138S, Cell Signaling Technologies, Danvers, MA, USA); Beta-Actin (3700S, Cell Signaling Technologies, Danvers, MA, USA), and CDT1 (ab70829, AbCam, Cambridge, UK); Lamin B1 (ab16048, AbCam, Cambridge, UK). Horseradish peroxidase conjugated secondary antibodies used in this studies were: goat anti-mouse IgG at 1:5000 dilution (31460, Invitrogen, Carlsbad, CA, USA), goat anti-rabbit IgG HRP conjugated (31430, Thermo Fisher Scientific, Asheville, NC, USA) at 1:1000, 1:2000, or 1:5000 dilutions. Blots were developed using Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and imaged using Molecular Imager^® Chemi doc™ XRS+ Imaging System (Bio-Rad, Hercules, CA, USA) aided by Image Lab Software Version 5.1 (Bio-Rad, Hercules, CA, USA).

The total proteins were prepared from cultured chondrocytes obtained as in ‘Quantitative analysis of gene expression by qRT‐PCR’ by lysis in the suspension of the RIPA Lysis and Extraction Buffer (Thermo Scientific) using Sonics Vibra Cell™ (Sonics & Materials, Newtown, CT, USA). Total cellular proteins were quantitatively analysed using the bicinchoninic acid (BCA) total protein quantitation assays 38. Equal proteins were separated by 8.0% SDS‐PAGE, blotted onto nitrocellulose membrane (Bio‐Rad, Hercules, CA, USA). The membranes were blocked by skim milk powder solution in Tris‐buffered saline and then incubated with diluted primary antibodies of CTNNB1 (AVIVA, San Diego, CA, USA) against β‐catenin, COL2A1 (Santa Cruz) against COL II by specifically binding to the C‐terminal epitope of a highly conserved motif between human and rabbit, and COL1A (COL‐1; Santa Cruz) against COL I, respectively, with the Monoclonal Anti‐β‐Actin (Sigma‐Aldrich) against the house‐keeping gene β‐actin used as an internal control. The binding proteins were detected with conjugated secondary antibody, goat anti‐rabbit IgG‐HRP (Santa Cruz) and visualized using Clarity™ Western ECL Substrate Kit (Bio‐Rad). The images were captured and analysed using the Image Lab (Beta 1) Version 3.0.1 Changelist 40296 software associated with Molecular Imager^®, ChemiDoc™ XRS+ Imaging System (Bio‐Rad).

Using PBS, BCa cells were washed 3 times and then were lysed for 30 minutes on ice with a solution that contained RIPA buffer, protease inhibitor and phosphatase inhibitor (Sigma‐Aldrich). The cell lysates were centrifuged at 13 000 × g for 15 minutes, and a Bradford protein assay (Bio‐Rad) was used to determine the protein concentration of the collected supernatants. Western blot analysis was performed after the total protein samples were separated by 7.5%‐15% SDS‐PAGE. Immunoreactive bands were visualized with an enhanced chemiluminescence kit (Bio‐Rad) and were then detected with a Molecular Imager ChemiDoc XRS + Imaging system (Bio‐Rad). Table S2 and Table S3 list the primary antibodies and secondary antibodies used, respectively.