Rna immunoprecipitation rip kit

The protein binding of Kcnq1ot1 and Ago2 was detected with a RNA Immunoprecipitation (RIP) kit (Millipore, Billerica, MA, United States). HK-2 cells were rinsed with pre-cooled PBS and supernatants were removed. Then, cells were lysed for 5 min with the same amount of RIPA lysis buffer and centrifuged for 10 min at 14,000 rpm at 4°C. The supernatants were coprecipitated with antibodies. A total of 50 μl magnetic beads used for each coprecipitation system was washed, resuspended with 100 μl RIP Wash Buffer, and then added with 5 μl of antibody for binding based on grouping design. The magnetic bead antibody complex was washed, followed by resuspension with 900 μl of RIP Wash Buffer and incubation with 100 μl of cell extracts overnight at 4°C. The sample was put on a magnetic stand to collect the magnetic bead antibody complex. RNA was extracted from samples after detachment using protease K for following RT-qPCR. Antibodies used in RIP assay were as follows: rabbit anti-Ago2 (ab186733, 1:50, Abcam) mixed for 30 min with rabbit anti-IgG (ab109489, 1:100, Abcam) as the negative control. The experiment was repeated three times.

The binding of LINC01296 to Argonaute-2 (AGO2) protein was detected using RNA immunoprecipitation (RIP) kit (Millipore Corp, Billerica, MA, U.S.A.). The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China). Part of the cell lysate was taken out as an input, and the other part was incubated with the antibody for coprecipitation. After being washed, the magnetic beads–antibody complex was resuspended in 900 μl RIP Wash Buffer and incubated with 100 μl cell lysate at 4°C overnight. Next, the sample was placed on the magnetic base to collect the magnetic beads–protein complex. RNA was extracted from the precipitated sample and input treated with proteinase K for subsequent PCR. Rabbit polyclonal antibody against AGO2 (ab32381, 1:10000, Abcam, Cambridge, U.K.) was used for RIPA with rabbit anti-human antibody against immunoglobulin G (IgG; ab6715, 1:1000, Abcam, Cambridge, U.K.) as an NC.

A RNA immunoprecipitation (RIP) kit (Millipore Corp.) was used to detect the binding between lncRNA XIST and Argonaute 3 (AGO2) protein. The RA cartilage cells were washed with pre‐cooled PBS, and the supernatant was discarded. An equal amount of RIPA lysis buffer (P0013B, Beyotime Biotechnology) was added into each sample and incubated in an ice bath for 5 minutes to lyse the cells. Subsequently, the cells were centrifuged, followed by the collection of the supernatant. A portion of cell extract was isolated as input, and the remaining part was incubated with antibody for co‐precipitation. Each system (50 μL) of co‐precipitation reaction was washed with magnetic beads and resuspended in 100 μL of a RIP Wash Buffer. The antibody (5 μg) was then added and incubated with the samples for binding. After being rinsed, the bead‐antibody complex was resuspended in 900 μL of RIP Wash Buffer and incubated with 100 μL of cell extract at 4°C overnight. The samples were then placed on a magnet base to collect the bead‐antibody complexes. The samples and input were, respectively, detached with protease K to extract RNA for subsequent PCR detection. The antibody used in RIP was rabbit anti‐human AGO2 (ab186733, 1:50; Abcam Inc.), which was incubated with samples at room temperature for 30 minutes. The rabbit anti‐human IgG (ab109489, 1:100; Abcam Inc.) served as NC.

Binding off lncRNA TUG1 to the USF1 transcription factor was investigated with the RNA immunoprecipitation (RIP) kit (Millipore, MA). Cells were pretreated with ice‐cold lysis buffer, centrifuged, and the supernatants harvested. Anti‐USF1 antibodies or negative control IgG (Abcam, Cambridge, MA; diluted 1:100) conjugated to magnetic beads in RIP Wash Buffer were then added to the supernatant to co‐precipitate RNA at 4°C overnight. Antibody‐conjugated beads with bound RNA were isolated, treated with proteinase K to digest proteins, and the RNA retained for further examination via PCR.