Bsa protein assay

Cells were lysed in ice cold 1% Triton lysis buffer [20 mM Tris–HCl pH 8.0, 10 mM EDTA, 1% Triton X-100 and 1× protease inhibitor cocktail (5892970001, Roche)] and protein concentration was measured using Bio-Rad BSA protein assay (5000116, Bio-Rad). Protein lysates were loaded on NuPAGE Tris–acetate 3–8% precast gels (EA03752BOX, Life Technologies) and ran at 150 V for 1.5 h. Gels were transferred onto PVDF membranes at 30 V for 1.5 h. Membranes were blocked in 5% milk in 0.2% Tween-20 in TBS for 1 h at room temperature and probed with primary antibodies against SORCS2 (1:750; AF4238, R&D Systems) and GAPDH (1:10,000; MAB374, Merck) diluted in blocking solution overnight at 4 °C. After washes (3 × 10 min) in 0.2% Tween-20 in TBS, membranes were incubated with secondary HRP-conjugated antibodies diluted 1:10,000 in blocking solution for 1 h at room temperature. After another three washes with TBS-0.2% Tween-20, blots were visualised using the Pierce ECL Plus Western Blotting Substrate (11527271, Thermo Scientific) and exposed using autoradiography film. Protein lysate obtained from HEK293 cells transfected with a plasmid overexpressing a human SORCS2 cDNA was used as a positive control.

Cell lysates from the above cell lines were prepared in RIPA buffer containing 1× protease cocktail inhibitors (Santa Cruz Biotechnology, Dallas, TX, USA), centrifuged at 12,000 rpm for 15 min at 4 °C. Protein concentrations were measured using the Bio-Rad BSA protein assay (Bio-Rad). Each sample’s protein (15 µg) was loaded into 10% SDS-PAGE gel and transferred onto a nitro-cellulose membrane. After blocking in 5% milk for 1 h, primary antibodies against NEK1 (27146-1-AP, Proteintech, Rosemont, IL, USA), NEK2 (D8, Santa Cruz Biotechnology), NEK3 (12843-1-AP, Proteintech), NEK6 (10378-1-AP, Proteintech), NEK7 (B5, Santa Cruz Biotechnology), and NEK9 (11192-1-AP, Proteintech) were incubated at 4 °C overnight. Secondary antibodies against mouse or rabbit with HRP were incubated for 1 h, then the membrane was developed using Immobilon Western Chemiluminescent HRP Substrate (MilliporeSigma, Burlington, MA, USA) and images were captured using Bio-Rad ChemiDoc XRS+ Imaging system. Band intensity was measured using ImageJ software (version 1.53k) and normalized to β-Actin of the same sample.

Infected cell lysates were collected for protein analysis, and cells were lysed in RIPA buffer (Cell Signaling) containing complete EDTA-free protease inhibitor (Roche) and phosphatase inhibitor cocktail (Roche). Protein concentrations were determined using a bovine serum albumin (BSA) protein assay (Bio-Rad). Cellular proteins separated by SDS–polyacrylamide gel electrophoresis were electrotransferred to nitrocellulose (NC) membranes and subjected to immunoblot analysis with various primary antibodies. Immunoblot analysis was done with various primary antibodies: anti-rabbit IRF3 (#4302; Cell Signaling), anti-rabbit pIRF3 (#29047; Cell Signaling), anti-rabbit cGAS (#31659; Cell Signaling), anti-rabbit STING (#29047; Cell Signaling), anti-rabbit Actin (sc-1616; Santa Cruz Biotechnology), and anti-rabbit GAPDH (sc-25778; Santa Cruz Biotechnology) antibodies.