Mab810r

HCMV Titan-infected cells were fixed 48 hours post-infection using 4% formaldehyde (Sigma). Fixed cells were blocked using 5% (w/v) BSA/PBS (PAA) and immunofluorescence was performed with mouse anti-cytomegalovirus antibody (MAB810R, Millipore), rabbit anti-US28 antibody kindly provided by Alberto Fraille Ramos [21 (link)], and the secondary anti-mouse Alexa Fluor^® 488-conjugated and anti-rabbit Alexa Fluor^® 568-conjugated antibodies. Imaging was carried out using a Olympus FSX-100 microscope.

The following antibodies were used: Anti-Cytomegalovirus, immediate early proteins (1:200 MAB810R, Millipore, clone 8B1.2, IgG2a) targeting the IE72 (IE1) and IE86 (IE2) proteins; Anti-Cytomegalovirus, late protein (1:200 MAB8127, Millipore, clone 1G5.2, IgG2a); Anti-Cytokeratin 20 (ready to use, IR777, Dako, Clone Ks20.8, IgG2a) was used as negative control.

All samples evaluated in this study were formalin-fixed, paraffin-embedded (FFPE) and as mentioned before, previously analyzed by an automated IHC staining method that we recently established in our research laboratory for detection of HCMV proteins [49 (link)].
Briefly, the slides were processed in Dako autostainer link 48 platform and consecutive sections were stained with antibodies for HCMV-IE (MAB810R, Merck Millipore Burlington, MA, USA), HCMV-LA (MAB8127, Merck Millipore) and CK20 (IR777, Dako A/S Glostrup, Denmark), the latter serving as negative control. The detailed immunohistochemical staining protocol is described in a previous publication [49 (link)].
Each slide was evaluated and scored for HCMV IE and LA protein positivity by an experienced professor and senior consultant in pathology at Akershus University Hospital. All slides were scored according to the percentage of HCMV positive cells: 0; <1%, 1; 1–24%, 2; 25–49%, 3; 50–74%, and 4; >75%. In our previous methodology study, a total of 111 breast cancer samples were stained. Two samples were from recurrent breast cancer from the same patients that underwent surgery for a second time, and were therefore excluded from further analyzes, given they were not suitable for survival and correlation analyses.

The following antibodies were used in immunoblot: anti-SAMHD1 rabbit polyclonal antibody (Ab) (Proteintech); anti-SAMHD1 rabbit polyclonal Ab specific for the phosphorylated T592 residue (ProSci); rabbit polyclonal anti-p85 subunit of PI3K (N-SH2 domain) and mouse monoclonal antibody (mAb) anti-IE1/IE2 viral proteins (MAB810R; Merck Millipore); mAb anti-UL97 (kindly provided by Thomas Mertens, Ulm University Medical Center, Germany); rabbit anti-Cdk1 (#77055) and mAb anti-tubulin (both from Cell Signaling); rabbit anti-Cdk2 (sc-163) and mAb anti-lamin A (C-3) (both from Santa Cruz). Horseradish peroxidase-conjugated secondary antibodies were from GE Healthcare. Alexa Fluor 488–conjugated mAb anti-IE1/IE2 viral proteins (MAB810X; Merck Millipore) or FITC-conjugated anti-Ki67 (SolA15; eBioscience) were used in flow cytometry. In type I IFN blocking experiments, a neutralizing anti-human IFN alpha/beta receptor chain 2 (CD118) mAb (MMHAR-2; Merck Millipore) was used at the concentration of 1 μg/ml, and freshly added at 2 dpi.

Mouse monoclonal antibody (MAb) 810R, which can detect both IE1 and IE2 proteins, and mouse MAb 6E1specific for IE1 were purchased from Chemicon and Vancouver Biotech, respectively. Mouse MAbs against IE1 (CH443), ICP36 (p52; UL44) and pp28 (UL99) were purchased from Virusys. Mouse MAb anti-β-actin and rat MAb anti-HA (3F10) conjugated with peroxidase were purchased from Sigma and Roche, respectively. Mouse MAbs for PML (PG-M13) and PKR and rabbit PAbs for STAT1 (E-23), STAT2 (C-20), IRF3 (FL425), and IRF9 (H-143) were purchased from Santa Cruz. Rabbit PAbs for p-STAT1 (Tyr701) and p-IRF3 (Ser396) were purchased from Cell Signaling. Rabbit PAb for p-STAT2 (Tyr689) and mouse MAb for HDAC1 were purchased from Upstate. Rabbit PAb for HDAC2 was obtained from Zymed. Mouse MAb for human IFNα/β receptor chain 2 (MAB1155) were purchased from Millipore. Anti-peptide rabbit PAb PML(C) specific for PML was described previously [23 (link)]. Mouse MAb anti-PML (5E10) [61 (link)] was kindly provided by Gary S Hayward (Johns Hopkins University School of Medicine, Baltimore, USA).

Mouse monoclonal antibody (MAb) 810R, which detects epitopes present in both IE1 and IE2, was purchased from Chemicon. Mouse MAbs against UL44 (p52) and UL99 (pp28) were obtained from Virusys. Anti-β-actin and anti-α-tubulin mouse MAbs were purchased from Sigma. Anti-HA rat MAb 3F10 and anti-myc mouse MAb 9E10, conjugated with peroxidase or labeled with fluorescein isothiocyanate (FITC), were purchased from Roche. Anti-ISG15 (F-9) and anti-STAT2 mouse MAbs were obtained from Santa Cruz. Mouse MAb against SRT epitope was previously described [91 (link)]. UBP43 antibody was previously described [92 (link)]. Rabbit polyclonal Ab (PAb) for STAT2 (C-20) and STAT2 phosphorylated at Tyr689 were purchased from Santa Cruz and Upstate, respectively. Rabbit PAb for ISG15 was kindly provided by Chin Ha Chung (Seoul National University, Seoul, Republic of Korea).