P2055

Cytosolic extracts from the hippocampus were prepared as described in the manufacturer’s instructions in the Nuclear and Cytoplasmic Extraction Kit (CW0199S, CoWin Biosciences). Cytoplasmic protein was divided into three groups: Input, DHX9, and IgG (negative control). The Input group was temporarily stored in a −80°C freezer and the remaining two groups were subjected to non-contact ultrasonic lysis, "M" strength, 5 min, 3 times. Then 1 μL rabbit IgG and 20 μL Protein A + G Agarose beads were added and the samples were incubated at 4°C for 2 h. After blocking, the supernatant was collected. Next, rabbit anti-DHX9 antibody or normal rabbit IgG antibody (1 μg, A7016, Beyotime) was added to the corresponding samples, which were then rotated at 4°C overnight. Next, protein A + G agarose gels (P2055, Beyotime) were added and the samples were incubated with rotation for 1 h at 4°C. The samples were washed three times with RIP wash buffer. Finally, RNA was eluted with TE buffer and extracted with Trizol (R401-01, Vazyme). Reverse transcription was followed by qPCR, and U6 RNAs was used as target reference control.

Lysis buffer (Beyotime, catalog P0013), which contained a protease inhibitor cocktail (MCE, catalog HY-K0010), was used for lysis. The indicated antibodies were used in cell lysates for immunoprecipitation at 4 °C overnight and then incubated with protein A/G (Beyotime, catalog P2055) at 4 °C for 3 h. Then the cell lysates were washed with lysis buffer four times and then analyzed by IB.

Cell lysis buffer for western or IP (P0013, Beyotime, Shanghai, China) was used to homogenize tissues or cells at 4 °C for 30 min. Lysates were centrifuged at 12 000× g for 10 min and then the supernatants were collected. Roughly 400 μg of total proteins in the volume of 200 μL were rotated with 4 μg anti−bodies at 4 °C overnight. The following day, 40 μL protein A + G agarose (P2055, Beyotime, Shanghai, China) was added and rotated at 4 °C for 6 h. The agarose beads were washed three times, re−suspended, and boiled in 40 μL SDS−loading buffer in sequence. The supernatants were collected for further examination.