Mini protean gel

Analysis of p-Syk and Syk levels were performed on 20 μg of total protein from samples processed in SDS-loading buffer and boiled for 10 min. Samples were loaded on a 4–20% Mini-PROTEAN gel (catalog no. 4561094; Bio-Rad) and transferred onto a polyvinylidene fluoride membrane. Membranes were blocked in 5% BSA–TBS with 0.02% Tween-20 for 1 h and incubated in primary antibody diluted (catalog no. 2710, 1:1,000; Cell Signaling; catalog no. 13198, 1:1,000; Cell Signaling) in 5% BSA–TBS with 0.02% Tween-20 overnight shaking at 4°C. The next day, after washing, secondary antibodies were applied for 1 h, shaking at RT. Membrane was washed three times for 5 min, developed using Pierce ECL Western Blotting Substrate (catalog no. 32106; Thermo Fisher Scientific), and imaged using a ChemiDoc imaging system. Blots were converted to grayscale and densitometry analysis was performed in ImageJ. Photoshop was used for post-quantification editing. Original unedited blots are available in the source data file for Fig. S3.

Whole cell lysates in 2x reducing sample buffer (125 mM Tris-HCl pH 6.8, 20% glycerol, 7.5% SDS, 5% 2-mercaptoethanol, 250 mM DTT, and 0.05% bromophenol blue) were denatured at 98° for 15 minutes, fractionated using SDS-PAGE (4–20% Mini-PROTEAN gel, Bio-Rad #4568093), and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore #IPVH00010). Immunoblots were probed with mouse anti-GFP (1:10,000 JL-8, Clontech #632380) and rabbit anti-β-actin (1:10,000 Cell Signaling #4967) followed by goat/sheep anti-mouse IgG IRDye 680 (1:10,000 LI-COR #926–68070) or goat anti-rabbit IgG-HRP (1:10,000 Jackson Labs # 111-035-144). HRP secondary antibody was visualized using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher # PI34095). Images were acquired using the LI-COR Odyssey Fc imaging system.

Protein was prepared from iPSC-CMs by direct lysis in ice-cold radioimmunoprecipitation assay buffer containing phosphatase and protease inhibitors (Santa Cruz Biotechnology) followed by collecting of cells using a cell scraper and mechanical trituration. After centrifugation (14,000 g at 4°C) for 20 min, the supernatant was collected. Protein content was determine using the Pierce BCA Protein Assay (Thermo Fisher Scientific). Protein was buffered in 4× Laemmli sample buffer (Bio-Rad) and boiled at 100°C for 5 min before electrophoresis. 10 μg of protein were electrophoresed on a 10% Mini-PROTEAN Gel (Bio-Rad) and transferred to a prewetted polyvinylidene fluoride membrane. After blocking and probing with a primary antibody overnight, proteins bands were visualized using an IRDye secondary antibody and visualized using the IR Odyssey imaging system (LI-COR). Total protein loading was further measured for standardization using the REVERT Total Protein Stain Kit (LI-COR). For ERK visualization, rabbit polyclonal phospho-ERK1/2 (T202/Y204, IB 1:500, 9101; Cell Signaling Technology), and mouse monoclonal ERK (clone 3A7, IB 1:500, 9107; Cell Signaling Technology) were used along with LI-COR IRDye 800 goat anti-rabbit (1:10,000) and 680 goat anti-mouse (1:10,000) secondary antibodies.