Np 40 lysis buffer

ES-2 cells were placed into 100 mm petri dishes, after treatment with erastin and YL-939-1 or DMSO for 1–10 h, cells were washed twice with PBS buffer, irradiated under an ultraviolet at 365 nm for 20 min on ice. Then, the cells were lysed with NP-40 lysis buffer (Beyotime) containing 1× protease inhibitor cocktail and 1 mM phosphatase inhibitor PMSF for 30 min. The proteins were collected by centrifugation for 15 min (16,000 × g, 4 °C), and then all protein concentrations were detected by a BCA protein quantification kit (Beyotime) and diluted to 1 mg/mL with NP-40 lysis buffer. Subsequently, click chemistry reaction reagents were added in following order: 50 μM TRAMA-N₃, 1 mM ascorbic acid, 100 μM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and 1 mM CuSO₄. The reaction was incubated for 1–2 h at room temperature in the dark with gentle shaking prior to addition of pre-chilled acetone (−20 °C). The proteins were collected by centrifugation (16,000 × g, 10 min, 4 °C), and washed twice with pre-chilled methanol. Eventually, the samples were dissolved in NP-40 lysis buffer, and added SDS-PAGE loading buffer before heated for 8 min at 100 °C, and loaded onto the SDS-PAGE gel. Then, protein lanes were visualized by in-gel fluorescence scanning (Typhoon FLA 9500) and coomassie blue staining.

Neuro2a cells were cultured in the presence or absence of 5 mM Hcy for 48 h, and then
washed with pre-cooled PBS. Phenylmethanesulfonylfluoride-containing NP-40 lysis
buffer (Biyuntian) was added and cells were incubated at 4°C with rotation for 30
min, then the supernatants were collected by centrifugation (10,000 g for 10 min).
Protein concentration was quantified with BCA and samples were stored at -20°C. Five
microliters of acetylated-lysine directed antibody (CST) was added to 500-1000 μg
total protein, mixed, and rotated overnight at 4°C. Then, 20 µL of 50% protein
A/G-agarose (Santa Cruz Biotechnology) was added, followed by rotation at 4°C for 2-4
h. After 5 min centrifugation at 5,000 g, the supernatant was removed by syringe.
Precipitates were washed with NP-40 lysis buffer 3 times, and supernatants were
discarded. Beads were re-suspended in 1× sodium dodecyl sulfate loading buffer,
denatured at 100°C for 5 min or at 60°C for 20 min. GAPDH was then detected by
western blot.

After 36 h of transfection, the cells were lysed with NP40 lysis buffer (Shanghai Biyuntian Biotechnology Co., Ltd.). We collected the lysate supernatant and divided it into two samples. Mouse anti-IgG (control group) and mouse anti-Flag monoclonal antibodies were added to the two samples at a ratio of 1:100 and incubated at 4°C for 24 h. Then, we added protein A and protein G to the samples at a ratio of 1:10 and incubated them at 4°C for 12 h. Finally, we discarded the supernatants of all samples and added 40 μL of PBS and 10 μL of 5× loading buffer. The subsequent steps were the same as those used for western blotting.

We transfected DEFs with pCMV-3C-HA, pCAGGS-IRF7-Flag, pCMV-HA, and poly(I:C) according to the appropriate experimental group and used NP40 lysis buffer (Shanghai Biyuntian Biotechnology Co., Ltd.) to collect cell samples 36 h after transfection. The samples were placed on ice for 30 min and centrifuged at 12,000 rpm for 10 min. The supernatants were aspirated into new EP tubes, the pellets were washed three times with PBS, and 1% SDS lysis buffer was added to lyse the cells on ice for 30 min, after which western blot detection was performed.

For protein extraction, 1 mL of NP-40 lysis buffer (Biyuntian) was transferred to each well of a six-well plate of microglia on ice for 30 min, and the cells were then centrifuged at 12,000 rpm for 10 min. The soluble protein solutions were then mixed with 5x sample buffer (Biyuntian) and boiled at 90^oC for 10 min. Equal amounts of protein (60 μg) from each sample were separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Sigma) and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% milk with Tris-buffered saline for 1 h, and then were incubated with the following primary antibodies at 4°C overnight: rabbit anti-iNOS, anti-Arg1 (1:500, Bioss) and rabbit anti-actin (1:1000, Santa Cruz Biotechnology). The membranes were washed three times in Tris-buffered saline with Tween and then incubated with the secondary antibody (peroxidase-conjugated IgG, 1:5000, Santa) at room temperature for 2 h. Binding antibodies were visualized using enhanced chemiluminescence on an imaging system (Amersham Imager 600).