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The oxidation site change between DVL3 wildtype and mutation

ABSTRACT: First of all, we transfect FLAG-PNPO and HA-DVL3 plasmids together into 293T cells. After 48h, we harvest them with IP lysate. First day, we used special antibody to connect with high-affinity magnetic beads overnight. Second day, the beads is cleanded and then connecte with protein IP lysate, whose connection is 2 mg/mL, overnight. Last day, we use elution buffer to elution binding protein and then conduct western blotting test. Next, we use Coomassie brilliant blue to stain the SDS-page gel two hours and then clean the gels with scrubbing solution overnight in roommate temperature. We cut the lanes and send to company to analysis the bingding protein with mass specture. Based on these rusults, We find different oxidation site between DVL3 wildtype and mutation.

ORGANISM(S): Homo Sapiens

SUBMITTER: Ye Yang

PROVIDER: PXD027698 | iProX | Wed Aug 04 00:00:00 BST 2021

REPOSITORIES: iProX

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Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP). Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4M-BM-0C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 M-NM-<l of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4M-BM-0C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.

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The oxidation site change between DVL3 wildtype and mutation

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