Ab32820

Drosophila S2 cell samples were boiled for 10′ in loading buffer separated on 6% (Spt6) or 10% (Co-IPs) SDS-PAGE gels and processed for western blotting using mouse α GFP (1:2000, Clone 71; D. van Essen), monoclonal mouse α Spt6 (25C6, Helmholtz Zentrum München, IgG2b 1:500) was raised against Spt6 amino acid residues 215–230 DYDDFSKYEEDDYEDD, rabbit α CENP-A (1:5000, ab10887, Abcam), mAB α Flag (Clone M2; 1:10000, F1804, Sigma-Aldrich), rabbit α H3 (1:10000, ab1791, Abcam) and mouse α tubulin AA4.3 (1:1000; DSHB). Secondary polyclonal Goat antibodies (Sigma) coupled to horseradish peroxidase Rabbit IgG HRP Linked Whole Ab, (NA934) and Mouse IgG HRP Linked Whole Ab (GE Healthcare, NA931) were used at 1:10000. HeLa cells were boiled in 2× Laemmli sample buffer and whole cell extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes using a Trans Blot System (BioRad). Antibodies used were SPT6 (Abcam, ab32820 rabbit polyclonal; 1:500), CENP-C (Gift from D. Cleveland UCSD, made in house from Covance clone #3024, rabbit polyclonal; 1:10000) and α-tubulin (Sigma, T9026 mouse monoclonal; 1:1000). Secondary antibodies used were Donkey anti-Rabbit 800 (Li-Cor, 926–32211) and Donkey anti-mouse 680 (Rockland, 610-744-124; both at 1:10000). Blots were visualized using Image Lab 6.1 (BIO RAD).

Click chemistry and immunofluorescence experiments were carried out after infection of Vero cells with KOS or EdC-labeled KOS as described [26 (link)]. To pulse label viral replication forks, 25 μM EdC was added to KOS infected cells at 4 hpi for 20 min before fixation with 3.7% paraformaldehyde. The Click-iT Alexa Fluor 488 Imaging Kit (ThermoFisher) was used to tag EdC labeled viral genomes and immunofluorescence was carried out using the following primary antibodies and dilutions: mouse anti-ICP8: ab20194 (Abcam), 1:200; mouse anti-PCNA: sc-056 (Santa Cruz), 1:200; mouse anti-MLH1: 550838 (BD Biosciences), 1:200; mouse anti-MSH2: Ab-2 NA27 (Calbiochem), 1:100; rabbit anti-MSH6: A300-023A (Bethyl Laboratories), 1:100; mouse anti-UL42: 2H4 ab19311 (Abcam), 1:200; mouse anti-ICP4: 58S, 1:500; mouse anti-Pol II (POLR2A): 4H8 (Abcam), 1:500; mouse anti-Sur2 (MED23): 550429 (BD Biosciences), 1:500; rabbit anti-Trap220 (MED1): sc-8998 (Santa Cruz), 1:250; rabbit anti-Spt5 (SUPT5H): A300-869A (Bethyl Laboratories), 1:200; and rabbit anti-Spt6 (SUPT6H): ab32820 (Abcam), 1:200. Goat anti-mouse and anti-rabbit alexa fluor 594-conjugated secondary antibodies (Santa Cruz) were diluted 1:500.

Paraffin-embedded, neoplastic, tumor tissue (8 µm; n = 24) and non-malignant control brain (8 µm; n = 10) sections were obtained from the Department of Clinical and Molecular Pathology, Palacky University and University Hospital Olomouc upon acquisition of a valid consent per the requirement of regional ethics committee and stained as described previously^{9 (link)}. In brief, following antigen retrieval, tissue sections were treated in 6% H₂O₂ to block endogenous peroxidase activity, then stained with primary antibody against SPT6 (1:100; ab32820, Abcam). After 1-h incubation at room temperature (RT), sections were washed, incubated with EnVision+ Dual Link System-HRP secondary antibody for 1 h, and immunoreactivity visualized using liquid DAB + substrate-chromogen system. Slides were washed, dehydrated through graded ethanol and mounted. The nuclei were counterstained with hematoxylin. SPT6 positivity was scored as negative (0), low (1), medium (2), and high (3).