Lambda protein phosphatase λppase

Nocodazole and Ku55933 were ordered from Selleckchem (Texas, USA). Lambda Protein phosphatase (λPPase) was bought from Sigma-Aldrich Corporation. The anti-ATM (1:1,000, ab201022, Abcam), anti-phospho-ATM (1:1,000, ab81292, Abcam), anti-Mad2 (1:1,000, ab70385, Abcam), anti-Mad1 (1:1,000, ab201022, Abcam), and anti-Cdc20 antibodies (1:1,000, ab183479, Abcam) were bought from Abcam (Cambridge, MA). The anti-HA (1:1,000, 3724T, Cell Signaling Technology), anti-Mad2 (1:1,000, 4636S, Cell Signaling Technology), and the HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). The anti-Flag antibody (1:1,000, F3165, Sigma) was purchased from Sigma. The anti-Mad2 antibody (1:1,000, YT2618, Immunoway) was bought from Immunoway. The anti-GFP (1:1,000, sc-9996, Santa Cruz), anti-Mad2 (1:1,000, sc-47747, Santa Cruz), anti-p-Thr (1:1,000, sc-5267, Santa Cruz), and anti-p-Ser antibodies (1:1,000, sc-81514, Santa Cruz) were bought from Santa Cruz Biotechnology. Goat anti-mouse lgG-H HRP (1:1,000, M21004L, Abmart) and goat anti-mouse lgG-L HRP (1:1,000, M21005S, Abmart) were purchased from Abmart.

RNA40 [5′-GCCCGUCUGUUGUGUGACUC-3′; at U5 region 108–127 nt of the HIV genome (accession no. NC_001802.1)] [14 (link)], RNA41 (a derivative of RNA40 in which adenosine replaces all uracil nucleotides), RNA649 [5′-GUCAGAGUGUGUACUUG-3′; position 24 649–24 665 nt in the SARS-CoV-2 genome (S2 spike protein region) (accession no. NC_045512.2)] [16 (link)], RNA649A (a derivative of RNA649 in which adenosine replaces all uracil nucleotides), IRS 661 (5′-TGCTTGCAAGCTTGCAAGCA-3′) [45 (link)] and ODN (5′-TCCTGCAGGTTAAGT-3′) [45 (link)] were synthesised by Integrated DNA Technologies. LyoVec (InvivoGen), a cationic lipid-based transfection reagent, was used to complex GU-rich ssRNA in a 2 : 1 (LyoVec:RNA) ratio as previously described [16 (link)]. RNA40 and RNA41 precomplexed with LyoVec were purchased from InvivoGen. Recombinant human ubiquitin specific peptidase 2 (USP2) and CU-CPT4a were purchased from R&D Systems. CU-CPT9a, JH-X-119-01, maraviroc, pacritinib (SB1518), plerixafor, and zimlovisertib were purchased from Selleck Chemicals. Lambda protein phosphatase (λ-PPase) and MG132 were purchased from Sigma-Aldrich.