Ab4566

The expansion microscopy technique [109 ] optimized for Arabidopsis seeds was conducted as previously described [73 (link)]. Anti-Fibrillarin antibody (ab4566, Abcam) and anti-alpha Tubulin antibody (ab89984, Abcam) were used in 1:500 dilution as primary antibodies. Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113, Abcam) and goat Anti-Chicken IgY H&L (Alexa Fluor® 555) (ab150170, Abcam) were used in 1:500 dilution as secondary antibodies. For each sample, a stack of nine images with 1-μm intervals were recorded by ZEISS LSM700 with 25× oil objective and ZEN software at 1024 × 1024 resolution in 8-bit. DAPI signals were excited by 405-nm laser and passed through SP490 filters. Alexa488 signals were excited by 488-nm laser and passed through BP490-635 filters. Alexa555 signals were excited by 555-nm laser and passed through 560–1000-nm filters. Pinhole sizes were kept as 1 airy unit for each color, and color channels were scanned separately. FIJI software was used for image processing and nuclear size quantification. Each stack of images was first Z-projected on maximum intensity and then the nuclear areas were determined based on DAPI signals. The zygotic nuclei were distinguished from the endosperm nuclei according to position and tubulin staining patterns.

Most common chemicals were purchased from Sigma Aldrich. MLN4924 (Takeda Pharmaceuticals), MLN7243 (Chemietek), MG132 (Viva Bioscience), Lipofectamine RNAiMAX (Invitrogen), siRNA On-TARGETplus SMARTpools (Dharmacon), protease Inhibitor Cocktail Tablets EDTA-free, Fugene6 HD (Roche), Suc-LLVY-AMC peptide (BostonBiochem). Rabbit monoclonal anti-NEDD8 (1:2000), Y297 (GeneTex, GTX61205), FK2 mouse anti-ubiquitin, stainings (1:250) (Viva Bioscience, VB2500), rabbit anti-ubiquitin (1:2000), western blotting (DAKO, Z0458), mouse anti-fibrilarin (1:1000) (ab4566), rabbit anti-nucleolin (1:1000) (ab22758), mouse anti-GAPDH (1:5000) (6C5, ab8245), rabbit anti-RPL7 (1:2000) (ab72550) (Abcam), mouse anti-tubulin (1:2000) (Cell Signalling, 3873), mouse anti-HA (1:2000) (12C5, 11583816001), mouse anti-GFP (1:500) (11814460001) (Roche), mouse anti-a6 proteasome subunit (1 μg/ml) (Enzo Life Sciences, BML-PW8100), rabbit polyclonal anti-HUWE1 (1:2000) (Bethyl laboratories, A300-486A), mouse monoclonal anti-p21 (1 μg/ml) (F-5, sc-6246, Santa Cruz), rabbit polyclonal anti-CDT1 (1:1000) (# 06-1295, Millipore), goat anti-mouse Alexa Fluor® 488 (115-545-146), goat Anti-Rabbit Alexa Fluor® 594 (111-585-008) (Jackson ImmunoResearch).

HeLa cells (ATCC^® CCL-2) were cultured in DMEM with high glucose, 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin (all from Millipore-Sigma, Burlington, MA, USA), in a humidified incubator with 5% CO₂ at 37 °C. For peptide treatment, 5 × 10⁵ cells were seeded in 6-well plates with 2 mL of full-DMEM medium 24 h before the treatment. Next day, the medium was exchanged with pre-warmed DMEM medium supplemented with respective peptides and incubated for defined times. As a negative control, HeLa cells were incubated with pre-warmed DMEM medium supplemented with 1× PBS without any of the CPPs.
Antibodies against fibrillarin (ab4566), coilin (ab87913), Sc35 (ab11826), tubulin (ab195883), 58K (ab27043) and M6PR (ab124767) were all purchased from Abcam (Cambridge, MA, USA). Alexa Fluor 555 Goat Anti-Rabbit (ab150078) and Alexa Fluor 488 Goat Anti-Mouse (ab150117), used as secondary antibodies, were also purchased from Abcam. Hoechst 43222 (H1399) were purchased from Invitrogen (Waltham, MA, USA).

NEIL1 antibodies were kindly provided by Dr. T. Rosenquist. Antibodies against TRIM26 (ab89290), NTH1 (ab70726), OGG1 (ab124741), and fibrillarin (ab4566) were from Abcam (Cambridge, UK). NEIL3 (sc-393703) and lamin a/c antibodies (sc-7292) were from Santa Cruz Biotechnology (Dallas, TX, USA), and tubulin antibodies (T6199) were from Merck (Gillingham, UK). Bacterial expression plasmids and protein purification of TRIM26, OGG1, NTH1, NEIL1, and NEIL3 proteins was performed as previously described [23 (link),24 (link),51 (link)].

The following antibodies were used: rabbit anti-Coilin (1:2000; ab210785, Abcam), mouse IgG1 anti-Fibrillarin (1:60; ab4566, Abcam), mouse IgG1 anti-nuclear pore complex proteins MAb414 (1:1000; MMS-120P-100, Eurogentec), rabbit anti-NPAT (1:700; A302–772A, Bethyl Laboratories), mouse IgG1 anti-PSPC1(1:100; clone IL4, SAB4200503, Sigma-Aldrich), rabbit anti-pSF3b155 (phosphorylated at Thr313; 1:400; clone D8D8V, 25009, Cell Signaling), rabbit anti-SF3b155 (1:600; clone D7L5T, 14434, Cell Signaling), mouse IgG1 anti-SMN1 (1:100; Survival of Motor Neurons, clone 2B1, 05–1532, EMD Millipore), mouse IgG1 anti-SRSF2/SC35 (1:400; ab11826, Abcam), mouse IgG1 anti-TDP-43/TARDBP (1:1500; clone 3H8, MABN45, EMD Millipore), and species-specific Alexa Fluor secondary antibodies (1:400; Thermo Fisher). A recent study^{45 (link)} proposed that the main target of the monoclonal SRSF2/SC35 antibodies is SRRM2 instead of SRSF2/SC35. However, SRRM2 is a spliceosome-associated protein that sharply localizes to nuclear speckles^{45 (link)}. We also confirmed that the monoclonal ab11826 antibody we used recognized SRS2-GFP⁺ speckles in fixed oocytes expressing SRSF2-GFP. The conclusions of our study are thus unaffected by the proposed SRSF2/SC35 antibody discrepancy^{45 (link)}.