Ab183136

We analyzed individually the elution profile of the contents of cells expressing EGFP-hnRNPDL isoforms, one isoform at a time. A total of 3 dishes of 10 cm per each hnRNPDL isoform transfection were pooled for SEC analysis. Experiments were performed in triplicate for each isoform. HeLa^DL-KO cells were transfected 24h after seeding with 3 μg DNA using Lipofectamine2000 (Invitrogen) and media was changed to fresh one after 5 h of transfection. Cells were collected 24 h post transfection, resuspended in 0.5 mL of 1xPBS supplemented with protease inhibitor cocktail (Roche) and lysed using 1 mL syringe (5-10 passes). Protein solubilization was confirmed by Bradford. Supernatant was collected by 5 min centrifugation at 1,000 g and 4°C, filtered with 0.45 μm PVDF membrane and loaded on a Superose 6 Increase 10/300 GL column (GE Healthcare) equilibrated with 1xPBS. The fractions were analyzed by Western Blot using a primary antibody against hnRNPDL (ab183136; Abcam) and the appropriate host-specific secondary antibody (IRDye 680RD Goat anti-Rabbit IgG (H + L), LI-COR, P/N 926-68071). The elution pattern of endogenous hnRNPDL in untransfected HeLa WT cells was also analyzed.

Transfected HeLa^DL-KO cells were stressed or not with 5 μg/ml actinomycin D for 3 h. Cells were then fixed with 4% parafolmaldehyde (Electron Microscopy Sciences, #15713-S), permeabilized with 0,2% Triton X-100 and blocked with 1% bovine serum albumin (BSA). Primary antibodies used were against G3BP (611127; BD Biosciences), nucleolin (sc-8031; Santa Cruz) and hnRNPDL (ab183136; Abcam). For visualization, the appropriate host-specific Alexa Fluor 488 or 555 (Molecular Probes) secondary antibodies were used. Slides were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen). Images were captured using a Leica TCS SP8 STED 3x confocal microscope (Leica Biosystems) with a 63x oil objective.

Gene sequences of hnRNPDL isoform 2 (DL2) WT, DL2 D259H, and DL2 D259N were synthesized and subcloned into the pUASTattB Drosophila expression vector (BioBasic Inc.). Flies carrying the transgenes were generated by performing a standard injection through the ϕC31 integrase-mediated transgenesis technique (BestGene Inc.). All Drosophila stocks were maintained in a 25°C incubator with a 12 h day/night cycle. Eye phenotypes were imaged by light microscopy. The w1118 line was used as control.
To prepare adult fly muscle for immunofluorescence, the Mhc-Gal4 driver was used to express the transgene in muscle at 25°C. Adult flies were embedded in a drop of OCT compound (Sakura Finetek) on a glass slide, frozen with liquid nitrogen and bisected sagitally by a razor blade. After fixing with 4% paraformaldehyde in PBS, hemithoraces were permeabilized with PBS containing 0.2% Triton X-100 and stained with anti-hnRNPDL antibody (ab183136; Abcam). Hemithoraces were additionally stained by Alexa Fluor 647 (Life Technologies) and DAPI according to manufacturer’s instructions. Stained hemi-thoraces were mounted in 80% glycerol, and the musculature was examined by STED (Leica SP8).