F1804 200ug

HEK293 cells in 10 cm² plates with 80% confluency were transfected with 10 µg smORF-3xFLAG constructs and 10 µg mock vector control 48 h prior to immunoprecipitation. Transfected cells were harvested in CelLytic M lysis buffer coupled with protease inhibitor cocktail (Sigma-Aldrich). Extracts were incubated at 4 °C overnight with 50 µl anti-FLAG M2 affinity gel (Sigma-Aldrich). The resulting immune complexes were washed, and the FLAG-tagged polypeptides were eluted by a competition with 3xFLAG peptide (Sigma-Aldrich) in wash buffer followed by western blot analysis with monoclonal anti-FLAG M2 antibody (1:1000, F1804-200UG, Sigma-Aldrich). After immunoprecipitation, proteins were separated on a pre-cast Tricine SDS-PAGE gel (Bio-Rad) and stained with GelCode Blue Stain reagent (Thermo Fisher Scientific). Gel slices were excised and digested with trypsin (Promega) overnight at 37 °C. Digested peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Ultimate 3000 system (Dionex) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Data were interrogated with smORF sequence by the Mascot Software (version 2.5.1) through Proteome Discoverer (Thermo Fisher Scientific)

Forty-eight hours post transfection, the HEK293 cells were lysed using CelLytic M lysis reagent (Sigma-Aldrich). Clarified cell lysates (30 μl) were mixed with 2× Tricine SDS Sample Buffer (Novex, Thermo Fisher Scientific) and run on 10–20% Tricine Protein Gels (Novex, Thermo Fisher Scientific) in Tricine SDS Running Buffer (Novex, Thermo Fisher Scientific) at 125 V for 90 min. Proteins were transferred to polyvinylidene fluoride membrane (0.2 μm, Bio-Rad) at 100 mA for 2 h in Tris-Gly Transfer Buffer (Novex, Thermo Fisher Scientific) supplement with methanol (Sigma-Aldrich). Immunoblots were incubated with primary monoclonal anti-FLAG M2 antibody (1:1000, F1804-200UG, Sigma-Aldrich) and anti-β-actin (1:10,000, AM4302, Ambion, Thermo Fisher Scientific) overnight at 4 °C and then secondary anti-mouse IgG and horseradish peroxidase-linked antibody (1:5000, Cell Signaling) at room temperature for 2 h. Immunoblots were developed with Western ECL (Clarity, Bio-Rad). Full, uncropped versions of all blot images are provided in Supplementary Fig. 16.

In these assays, HEK and U2OS cells were transfected for 48 h with 3xFLAG-dyskerin plasmid (Addgene), as described above and then transferred in RIPA lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA, 1% NP40) supplemented with anti-phosphatase and anti-protease (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, Sigma-Aldrich, Merk, Rahway, NJ, USA). Lysates of transfected cells (about 1 mg) were added to 20 μL of TBS 1x-rinsed beads (anti-FLAG M2 Affinity Gel A2220-SIGMA ALDRICH) and incubated overnight at 4 °C. Following a 1 min spin at 8000× g at 4 °C, unbound proteins were removed by rinsing beads with 200 μL of TBS 1x wash buffer three times. Pulled-down proteins were then analyzed by immunoblotting. The experiments were repeated three times independently. The primary antibodies used were anti-CYPA/PPIA (104698, Genetex); anti-PRDXII (Ab133481, ABCAM); anti-Zo-1 (bs1329, Bioss); anti-GAR1 (PA5-63656, Thermofisher); and anti-FLAG (F1804–200UG, Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were anti-Rabbit (A120-100P) and anti-Mouse (A90-137P), Bethyl Laboratories, Montgomery, AL, USA.