Complete protease inhibitors and phosstop

Cell pellets were resuspended in SDS lysis buffer (50 mM Tris, pH 6.8, 2% SDS, 5% glycerol, two mM DTT, 2.5 mM EDTA, 2.5 mM EGTA, 4 mM NaVO₄, and 20 mM NaF complemented with Complete Protease inhibitors and PhosStop [Roche]) and sonicated for 10 s with 2 s ON and 2 s OFF. After 5 min at 95°C and centrifugation for 5 min at 10,000g, supernatant proteins were quantified using the tryptophan fluorescence method (Wisniewski and Gaugaz (2015) Anal Chem 87: 4110–4116). 1.2 μg of protein extracts were subjected to the capillary-based immunoassay Jess system (Protein Simple) using the 12–230 kD separation module according to the manufacturer’s instructions. Anti-STING (13647S), anti-cGAS (15102), anti–NF-κB p65 (6956), anti–phospho-NF-κB p65 (3033), anti–phospho-STING (Ser366) (19781), anti-TBK1 (3504), anti–phospho-TBK1 (5483), and anti–pan-Actin (4968) antibodies were purchased from Cell Signaling Technology and diluted 40-fold in the AD2 blocking buffer before use.

Yeast growth
in (Yeast Extract–Peptone–Dextrose)
YPD Broth is meticulously cultivated using a well-defined procedure.
To begin, 50 g of YPD Broth was blended with 1 L of distilled water,
ensuring a precise mixture. This suspension underwent autoclaving
at 121 °C for a duration of 15 min. Following this, yeast cultures
are introduced into detergent-free containers. A brief vortexing was
then carried out to uniformly disperse the yeast cells throughout
the medium. The yeast cultures were subsequently nurtured in a shaking
incubator at 300 rpm.
After yeast cell collection and cleanup
with a PBS, 5 g of yeast cells was suspended in the lysis buffer containing
8 M urea, complete protease inhibitors and PhosSTOP (Roche), and 100
mM ammonium bicarbonate (pH 8.0), followed by incubation on ice for
30 min with periodical vortexing. The cells were lysed for 3 min using
a homogenizer (Fisher Scientific) and then sonicated under a 50% duty
cycle, level 10 output for 20 min on ice with a Branson Sonifier 250
(VWR Scientific). The yeast lysate was centrifuged at 14,000g for 10 min at 4 °C to collect the supernatant containing
extracted proteins. The concentration of total proteins was measured
by a bicinchoninic acid (BCA) kit (Fisher Scientific) according to
the manufacturer’s instructions, and the sample was stored
at −80 °C.

Whole cell extracts were obtained by solubilizing cells in lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), plus Complete protease inhibitors and PhosSTOP (Roche Diagnostics)). Lysates were clarified by centrifugation at 16,000g for 15 min.