Ip co ip kit

Co‐IP was carried out utilizing an IP/Co‐IP Kit (Thermo Fisher Scientific) as per the manufacturer's instructions. Initially, cellular lysates comprising 500 μg of protein were mixed with 5 μg of IP antibody per sample in a microcentrifuge tube and incubated for 12 h at 4°C. Subsequently, the antigen/antibody complex was allowed to attach to Protein A/G magnetic beads for 1 h at 37°C. The beads were washed twice with an IP Lysis/Wash Buffer and once with purified water. The protein was collected and used for western blotting, and the Clean‐Blot IP (Thermo Fisher Scientific) was utilized to prevent interference from the heavy chain and light chain segments of the antibody following the protocol.

An RIP Kit (Millipore, Burlington, MA, USA) was applied in this study. In brief, 50 μL of a magnetic beads suspension was washed and resuspended in 100 μL of RIP wash buffer. Then, 5 μg of anti‐DDX3 antibodies were added into each tube and incubated with rotation for 30 min. One hundred microliter lysates were added to each beads‐antibody complex in RIP immunoprecipitation buffer and all the tubes were incubated while rotating overnight at 4°C. The purified RNA was analyzed using RT‐PCR or qRT‐PCR.
An IP/Co‐IP Kit (#88828, ThermoFisher) and Co‐IP Kit (#26149, ThermoFisher) were used to determine the interaction between DDX3 and YY1. For DDX3 immunoprecipitation, DDX3 antibody was immobilized on AminoLink Plus Coupling Resin, and then incubated with cell lysates overnight at 4°C. After elution of the immunoprecipitation products, the products were boiled for 10 min with 5X Lane Marker Sample Buffer for the next analysis. For YY1 immunoprecipitation, the whole lysates were incubated with the beads‐antibody complex. After the products were washed with lysis buffer, they were boiled for 10 min with 1 × SDS loading buffer for the next analysis. The primary antibodies used are listed in Table S7.

Purified metacyclic forms were lysed with PBS containing 0.5% nonionic detergent Igepal CA630 (USB Corporation), and cleared lysates were used for immunoprecipitation. A/G magnetic beads (Pierce cross-link magnetic immunoprecipitation/coimmunoprecipitation [IP/co-IP] kit) cross-linked with MAb 1G7, according to the manufacturer's instructions (Thermo Scientific), were incubated for 1 h with parasite lysates at room temperature under constant agitation. Following washes with PBS containing 0.25% Igepal CA630, the bound proteins were eluted with the elution buffer, pH 2, and subjected to SDS-PAGE followed by Western blotting using MAb 1G7 or MAb 5E7. Western blot analysis for detection of LAMP2 protein was performed with detergent extract of HeLa cells incubated under different conditions.