Ibright fl1500

Plasma was diluted 1/100 in H₂O. Two volumes of this sample were mixed with one volume of NuPAGE® LDS sample buffer (4X) (Thermofisher) containing reducing agent (DTT 0.33 M) and then denatured at 90°C for 10 min. Proteins were separated in NuPAGE 10% Bis-Tris gel (Thermofisher). The proteins were transferred onto a nitrocellulose membrane using iBlot (Invitrogen). The membranes were incubated overnight with primary antibody (Goat IgG fraction anti-mouse complement C3, MP BIOMEDICALS, #55463), followed by a secondary antibody (rabbit anti-goat HRP, Thermofisher, #31402). Revelation was done by chemiluminescence using a substrate for HRP (SuperSignal® WestDuraLuminol Thermofisher, #1856145), detected by iBright Western Blot Imaging System (iBright FL1500 Thermofisher).

LV tissue samples were ground in liquid nitrogen and sonicated in PBS with 1X phosphatase and protease inhibitors. The protein content in the sample was quantified using the BCA protein assay kit (ThermoFisher Scientific, 23225). Then, 40µg protein was loaded on each well and separated using SDS-PAGE and transferred into methanol-activated PVDF membrane. The membrane was then blocked with 5% non-fat milk in TBST for 1 hour. Then, the membrane was incubated with primary antibodies for cardiac β-myosin heavy chain (Abcam, ab50967 at 1:1000 dilution), atrial natriuretic peptide (Thermo Fisher Scientific, PA5-29559 at 1:1000 dilution), and GAPDH (Cell Signaling Technologies Inc., CST 97166 at 1:1000 dilution) overnight at 4°C. Next day, the membrane was washed with TBST three times, 10 minutes each. The membrane was then incubated with HRP-conjugated secondary antibodies (Abcam ab97064, ab97030 at 1:4000 dilution in 5% non-fat milk in TBST) for 1 hour at room temperature. Then, the membrane was washed with TBST three times, 10 minutes each to remove unbound secondary antibodies. The protein band was detected using an iBright FL1500 instrument (Thermo Fisher Scientific) and the bands were quantified using ImageJ analysis software from the National Institutes of Health.

hDPCs (1 × 10⁶ cells/dish) were seeded in 100 mm culture dishes and cultured for 24 h. D-panthenol was treated at appropriate concentrations for 24 h. Cells were then washed with ice-cold PBS and lysed on ice in an M-PER buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with a Complete™ protease inhibitor cocktail and phosphatase inhibitor (Roche, Indianapolis, IN, USA). 40 μg of protein was analyzed by Western blotting with appropriate antibodies to evaluate protein expression; β-catenin (1000:1 dilution, Santa Cruz, CA, USA), CDKN2A/p16INK4a (1000:1 dilution, Abcam, Cambridge, UK), p21 (1000:1 dilution, cell signaling technology, Danvers, MA, USA), caspase3 (1000:1 dilution, cell signaling technology, Danvers, MA, USA), GAPDH (2000:1 dilution, Santa Cruz, CA, USA). Western blot was analyzed by a chemiluminescence detector (iBright FL1500, Thermo Fisher Scientific, Waltham, MA, USA).