Irdye 800cw

Purified CUL5/Rbx2, NAE1/UBA3, UBE2F, and VCBC were mixed at a final concentration of 5 μM each, with 10 μM NEDD8 and 1 mM of freshly prepared ATP in an assay buffer, 25 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, and 0.01% Tween 20. The mixture was incubated at 20 °C for at least 1 h. NEDDylation of CUL5 was verified by PAGE analysis. A 10-μL aliquot of the resulting mixture was mixed with 1 μM sA3G or its mutant, 5 μM E2L3, 30 μM ubiquitin or its mutant, 20 μM DNA or RNA oligomer, and an additional 1 mM ATP, and the total volume was adjusted to 50 μL with assay buffer. Finally, 0.2 μM UBE1 (Sigma Aldrich) were added. The mixture was incubated at 20 °C, and an aliquot of the reactant was harvested for further analysis after 0, 7.5, 15, and 30 min.
To detect sA3G, its mutants and their ubiquitinated variants selectively, a c-myc-tag sequence was attached at their C-termini and detected by western blotting with primary (monoclonal anti-c-myc tag, 9E11, Abcam) and secondary antibodies (monoclonal anti-mouse IgG, ab216772, conjugated with a fluorophore IRDye 800CW, Abcam). Protein bands were detected with a ChemiDoc system (Bio-Rad Laboratories) and analyzed using NIH ImageJ^{59 (link)}.

Organoids were harvested and washed twice with Advanced DMEM/F12 and twice with PBS, before organoids were re‐suspended in 100–200 μl of RIPA buffer with protease inhibitor (1x, Thermo Fisher Scientific, 78440) added. Organoid suspension was incubated for 30 min on ice, with strong vortex every 5 min. Cell pieces and debris were removed by centrifugation at 21,300 g. Supernatant was harvested. Protein concentration was measured by BCA assay (Thermo Fisher Scientific). Equal amount of each protein sample was mixed with Sample Buffer (Bio‐rad) and beta‐mercaptoethanol (Merck, M6250) according to the manufacturer's protocol. Mixture was heated at 95°C for 5 min and cooled down to room temperature.
Samples were separated on a 4–12% SDS–PAGE and transferred to nitrocellulose membranes. Proteins were detected by incubation with primary antibodies, SOX9 (1: 1,000, Merck, AB5535) and β‐Actin (1: 5,000, Merck, A3854) at 4°C overnight and subsequently secondary antibodies, donkey anti‐Mouse IRDye 800CW (1: 1,000, AbCam, ab216774) and donkey anti‐Rabbit IRDye 680RD (1: 1,000, AbCam, ab216779). Protein bands were visualised using Li‐Cor Odyssey system.

Pseudoviruses were concentrated by ultracentrifugation before being reduced in Laemmlli buffer with 10% β-mercaptoethanol. Proteins were transferred to a nitrocellulose membrane and were probed overnight with a polyclonal rabbit anti-SARS spike protein (NOVUS; NB100-56578) and mouse anti-p24 (abcam; ab9071), followed by 1 hour incubation with the secondary antibodies, IRDye 680RD Goat anti-mouse (abcam; ab216776) and IRDye 800CW Goat anti-rabbit (abcam; ab216773). Fluorescence was measured using the Odyssey Imaging System (LI-COR Biosciences).

Antibodies used for ChIP were H3K9me3 (ab8898, Abcam), H3K4me2 (ab7766, Abcam and #39141, Active Motif), H3K27me3 (#39155, Active Motif), H3K27Ac (ab4729, Abcam) and KDM1/LSD1 (ab17721, Abcam). Primary antibodies used for immunodetection after Western Blot were TetR monoclonal antibody 9G9 (#631131, TAKARA), DDX19A (orb242165, Biorbyt or ab108462, Abcam), RNA:DNA Hybrid Antibody, clone S9.6 (MABE1095, MERCK/Sigma-Aldrich) and KDM1A (#61607 and #39186, Active Motif). Secondary antibody used for immunofluorescence was the Goat Anti-Mouse IgG H&L (Alexa Fluor® 594, ab150116, Abcam). Antibody used for detection of DDX19A-GST was the goat anti-GST antibody (GE Healthcare, #27–4577-01). Secondary antibodies for analysis of Western Blots were either coupled to horseradish peroxidase (GE Healthcare) or to IRDye^® 800CW (ab216773, Abcam).

To visualize protein expression during mini-genome assays, around 500,000 transfected cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris [pH 7.4]) supplemented with an EDTA-free protease inhibitor cocktail tablet (Roche).
Proteins were detected with mouse α-FLAG (F1804, Sigma), rabbit α-Vinculin (AB129002">AB129002, Abcam), rabbit α-PB2 (GTX125926, GeneTex), and mouse α-NP ([C43] AB128193, Abcam). The following near-infrared (NIR) fluorescently tagged secondary antibodies were used: IRDye 680RD goat anti-rabbit (IgG) secondary antibody (AB216777, Abcam) and IRDye 800CW goat anti-mouse (IgG) secondary antibody (AB216772, Abcam). Western blots were visualized using an Odyssey Imaging System (LI-COR Biosciences).