Peg3_tRF_mimics

Description

To confirm a direct role for 3’-tRFs in silencing of endogenous Peg3, we transfected mouse embryonic fibroblasts with synthetic 3’-tRF mimics and observed downregulation of PEG3 protein specific to Arg-TCT tRF3b. Samples were loaded in triplicate left-to-right, using the following 3'-tRF mimics: i) NT control (/5Phos/rUrArCrGrGrArCrUrUrArArGrCrGrGrCrUrArCrArUrA); ii) Phe-GAA tRF3b (/5Phos/rUrCrGrArUrCrCrCrGrGrGrUrUrUrCrGrGrCrArCrCrA); iii) Asn-GTT tRF3b (/5Phos/rUrUrGrArArCrCrCrArCrCrCrArGrArGrGrCrGrCrCrA); iv) Arg-TCT tRF3b (/5Phos/rUrCrGrArGrUrCrCrCrGrGrCrGrGrArGrUrCrGrCrCrA).

Steps to reproduce

Mouse embryonic fibroblasts (ATCC; SCRC-1008; 200,000 cells per well) were seeded in 6-well plates one day prior to transfection with 187 pmol synthetic 3’-tRF mimics (Integrated DNA technologies) using Lipofectamine RNAiMAX (Invitrogen; 13778075). Protein was harvested 48 hours post-transfection. Cells were washed with ice-cold PBS pH 7.2 (Gibco; 20012027) and lysed in ice-cold RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM Tris-Cl pH 8.0) with 1X cOmplete mini EDTA-free protease inhibitor (Roche; 11836170001). Cells were scraped from the dish surface and sonicated for 5 minutes in 30 second on/off cycles. Cell debris was pelleted by centrifuging at 18,000 rcf for 10 minutes at 4°C. Proteins were precipitated from the supernatant in 80% acetone, and resuspended in a minimum volume of 1X reducing agent-free Laemmli buffer (50 mM Tris-Cl pH 6.8, 10% glycerol, 1% SDS). Total protein was quantified using the Pierce BCA protein assay (Thermo Scientific; 23225). DTT (50 mM) and bromophenol blue (0.025% w/v) were added to the lysates before they were heated for 5 minutes at 95°C and centrifuged at 18,000 rcf for 5 minutes at 4°C. Proteins were separated by 7.5% SDS-PAGE and transferred onto a 0.45 μM nitrocellulose membrane (Bio-Rad; 1620115) using the Mini Trans-Blot system (Bio-Rad; 1703930). Transfer efficiency was checked by staining with Ponceau S (0.1% w/v) in acetic acid (5% v/v). Membranes were blocked in 5% Blotto non-fat dry milk (Santa Cruz Biotechnology; sc-2324) in TBS with 0.1% Tween 20 (Thermo Scientific; J20605-AP) for a minimum of 1 hour. All antibodies were diluted in the same buffer, used as follows: anti-PEG3 (Thermo Scientific; PA5-99683) 1:500 overnight at 4°C; anti-tubulin (Abcam; ab6046) 1:1000 for 1 hour at room temperature; anti-rabbit-HRP (Jackson Immuno Research; 111-035-144) 1:3000 for 1 hour at room temperature. Blots were developed using Pierce ECL Western Blotting Substrate (Thermo Scientific; 32106) and an Odyssey FC Imaging System (LICORbio).

Institutions

• Cold Spring Harbor Laboratory

  NY, Cold Spring Harbor

Categories

Funders

• National Institutes of Health

  United States Department of Health and Human Services

  Maryland, United States

  Grant ID: GM138669

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