Cell lysis buffer

The brains from healthy mice and mice injected with MM5 cells were harvested at 9 and 14 days post injection (early growth phase), minced with a scalpel and snap frozen in liquid nitrogen. Half of the frozen tissue from each sample was homogenized and lysed in 1 ml RayBio Cell lysis buffer supplemented with protease inhibitor cocktail (Roche). After centrifugation, the clear supernatants were analyzed on RayBiotech Biotin Label-based Antibody Array 1 (detecting 507 proteins) according to manufacturer instructions followed by image acquisition and data processing as described in Supplementary Information (SI).

HDPCs (5 × 10⁵) were seeded in 100 mm dishes and incubated at 37°C for 24 h. Then, cells were treated with PG (0-10 μM) in the presence or absence of 20 μM LY294002, 40 μM CHX, 5 μM CHIR99021, 0.1 mM H₂O₂, or 2 μM MG132 at 37°C. The drug-treated cells were lysed using cell lysis buffer (#4719964001; Roche; Merch KGaA) containing phosphatase inhibitor cocktail (#4906845001; Roche; Merch KGaA). The protein concentration was quantified using Pierce BCA Protein Assay Kit (#23225; Thermo Fisher Scientific). Twenty micrograms of protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes (#88018; Thermo Fisher Scientific). The membranes were incubated with the corresponding primary antibodies overnight at 4°C, and then horseradish peroxidase-conjugated anti-mouse IgG (#7076S; Cell Signaling Technology; CST; USA) or anti-rabbit IgG (#7074S; CST) secondary antibodies were incubated for 2 h at room temperature. The blots were detected using Clarity Western ECL substrates (#1705061; Bio-Rad Laboratories, USA) and the intensities of the protein bands were analyzed with ImageJ software version 1.53t (National Institutes of Health). The following primary antibodies and dilution are listed in Table 2.

Cells were harvested with trypsin, pelleted by centrifugation at 4 °C and disrupted in cell lysis buffer (Roche) on ice for 15 min. The protein concentration was determined with a BCA Protein Assay Kit (Thermo) as described in the manufacturer’s manual. Protein lysate (50 μg) was subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene difluoride membrane. After blotting, the membrane was incubated with a specific primary antibody (EHD1: Proteintech; GAPDH: ZSGB-BIO) at 4 °C overnight. After washing with TBST (TBS containing 0.05 % Tween 20), the membrane was incubated with secondary antibodies (1:10000; ZSGB-BIO) for 1 h at room temperature. Protein bands were visualized using a Super ECL Reagent (HaiGene), and the protein expression was quantified by densitometry.

Adult mouse heart was dissected and homogenized by 10 strikes with a polytron probe in ice-cold RIPA buffer (Roche). Cell extracts were prepared by solubilizing 10⁷ cells in 1 ml of cell lysis buffer (Roche) for 10 min at 4 °C. After brief sonication, the homogenates or cell lysates were transferred to a 1.5 ml tube and rotated for 1 hour at 4 °C, then centrifuged for 10 min at 10,000 g at 4 °C and the supernatant was transferred to a new tube for protein quantification and immunoprecipitation. Immuno-precipitations were performed with Pierce direct IP kit (Thermo Fisher Scientific Inc., #26148), which immobilized 25 μg VCL (Invitrogen, #MA5–11690) or 25 μg SCN5A (Invitrogen, #PA5-34190) antibodies on agarose-resin support directly to improve specificity. Homogenate (1.0 mg/reaction) was mixed with immobilized antibody-agarose resin complex at 4 °C overnight. After washing to remove non-bound (presumably undesired) components of the sample, the antigen is recovered by dissociation from the antibody-agarose resin complex with elution buffer supplied in the kit. The immunoprecipitated samples were analyzed by Western blotting by probing with anti-SCN5A (Sigma, #SAB2107930) or anti-VCL (Sigma, #V4139).

Pathway analysis was performed as per the manufacturer’s instructions of the PathScan^® Intracellular Signaling Array Kit (Fluorescent Readout) (#7744; Cell Signaling Technology, EuroClone, Milan, Italy). Briefly, 24 h serum-starved MES (1.8 × 10⁶ cells) were left to adhere to HA, or HA-bound-C1q, as described above for the indicated periods of time at 37°C. Then, the cells were washed with ice-cold 1× PBS and lysed in 1× ice-cold Cell Lysis buffer containing a cocktail of protease inhibitors (Roche Diagnostics). The Array Blocking Buffer was added to each well and incubated for 15 min at RT. Subsequently, an equal amount of total lysate (0.8 mg/ml) was added to each well and incubated for 2 h at RT. After washing, the biotinylated detection antibody cocktail was added to each well and incubated for 1 h at RT. Streptavidin-conjugated DyLight 680 was added to each well and incubated for 30 min at RT. Fluorescence readout was acquired using the LI-COR Biosciences Infrared Odyssey imaging system (Millennium science) and data processed by the software Image studio 5.0.