A32959

Protein lysates from tumor tissue and cultured tumor cells were made using RIPA buffer (8990, Thermo Fisher Scientific) containing protease and phosphatase inhibitors (88265 and A32959, respectively, Thermo Fisher Scientific). The following antibodies were used for immunoblotting: STING (13647S, Cell Signaling Technology), TBK1 (3504T, Cell Signaling Technology), p-TBK1 (s172) (5483T, Cell Signaling Technology), IRF3 (MA5-32348, Invitrogen, Thermo Fisher Scientific), p-IRF3 (4947S, Cell Signaling Technology), NF-κB (ab16502, Abcam), p–NF-κB (3033S, Cell Signaling Technology), PD-L1 (ab213480, Abcam, 66248-1-Ig, Proteintech), GAPDH (SC-32233, Santa Cruz Biotechnology), vinculin (4650S, Cell Signaling Technology), Foxp3 (ab215206, Abcam), and CTLA-4 (BE0164, Bio X Cell).

Protein was isolated from bisected testes of young (3 months, n = 2) and old AROM⁺ animals (7 months, n = 2) and the corresponding WT littermates (n = 2, each) by disruption of the tissue using lysing tubes containing ceramic beads (Lying Matrix D; MP BiomedicalsTM; Irvine, CA, USA) and RIPA buffer supplemented with protease and phosphatase inhibitors (A32959; Thermo Fisher Scientific Inc., Waltham, MA, USA) in adequate volumes in a tissue homogenizer (FastPrep-24TM 5G; MP BiomedicalsTM; Irvine, CA, USA) with 2–3 cycles at 6.0 m/s speed for 120 s with pauses of 2 min on ice, followed by sonification and centrifugation at 13,000 rpm for 15 min. The sample concentration was measured by Lowry assay (DCTM Protein Assay; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and equal amounts of protein (10 µg/lane) were loaded. The membrane was incubated with a polyclonal TRPV2 antibody (1: 600; HPA044993; Atlas Antibodies, Stockholm, Sweden) at 4 °C over-night and a monoclonal αTubulin antibody (1: 10,000; ab52866; Abcam, Cambridge, UK) for 1 h at room temperature, both produced in rabbit. An IRDye 800CW secondary antibody donkey-α-rabbit (1: 10,000; 926-32213; Li-COR Biosciences, Lincoln, NE, USA) was used and bands were detected with the Odyssey CLx imaging system (Li-COR Biosciences, Lincoln, NE, USA). Intensities were measured, background subtracted and normalized to αTubulin.

Proteins from frozen mouse hearts were prepared by lysis in ice-cold RIPA buffer (89900, ThermoFisher Scientific) containing protease and phosphatase inhibitor (A32959, ThermoFisher Scientific). Tissue was homogenized using a bullet blender bead lysis kit (Next Advance), and protein concentrations were determined with a Pierce BCA Protein Assay Kit (23225, ThermoFisher Scientific). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-15% gradient gel (4561086, Bio-Rad) and transferred to PVDF membrane using an iBlot 2 transfer system (ThermoFisher Scientific). Protein expression was measured by chemiluminescence using a ChemiDoc imaging system (Bio-Rad). Proteins were detected with the following primary antibodies: anti-caspase-1(p20) (AdipoGen; 1:500), anti-β-actin (ab8226, Abcam; 1:1000) and anti-GAPDH (sc-365062, Santa Cruz Biotechnology; 1:1000).

Lysis of cells and EVs was performed using the radioimmunoprecipitation assay (RIPA) buffer (89900, Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (A32959, Thermo Fisher Scientific). An equal amount of proteins were run on SDS gels under denaturing conditions and blotted onto nitrocellulose membranes. The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-CD99 (sc-53148; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Ezrin (E8897; Sigma-Aldrich); anti-XAGE1A (A61802; Epigentek, Farmingdale, NY); anti-GAPDH (5174; Cell Signaling); anti-GPI (MA515396; Thermo Fisher Scientific), anti-IGFALS (sc-377131; Santa Cruz Biotechnology).