*2.8. Immunoprecipitations*

PBMC-derived monocytes for endogenous IPs were lysed using 1 X lysis buffer (150 mM NaCl, 50 mM TrisHCl (pH 8.0), 1 mM EDTA, 1% NP40) and supplemented with EDTA-free Complete Mini protease Inhibitor Cocktail Tablets as well as a PhosSTOP phosphatase-inhibitor cocktail from Roche, with 50 mM NaF and 2 mM Na3VO3 (Sigma, Merck, Darmstadt, Germany). Immunoprecipitations (IPs) were carried out on rotator at +4 ◦C for 4 h by co-incubation of the lysates from the stimulated cells (400–500 μg of protein/IP) with specific anti-TIRAP antibodies covalently coupled to Dynabeads (M-270 Epoxy, Thermo Fisher Scientific, Waltham, MA, USA), as suggested by the manufacturer, followed by extensive washing of the beads in a lysis buffer. Co-precipitated complexes were eluted by heating the samples in a 1× loading buffer (LDS, Invitrogen), without reducing the reagen<sup>t</sup> to minimize the antibodies' leakage to the eluates. Eluates were transferred to clean tubes, followed by the addition of DTT to the 40 mM concentration, heating, and Western blot analysis.

### *2.9. Immunofluorescence and ScanˆR Analysis*

Monocytes were isolated from PBMC using CD14 MicroBeads UltraPure (Miltenyi Biotech) and seeded in 96-well glass-bottom plates (P96-1.5H-N, Cellvis, CA, USA) at 50 K cells/well and in 24-well culture plates (250 K cells/well). Monocytes were differentiated into MDMs and transfected two times with a *TIRAP* siRNA and AllStar siRNA control, using the standardized protocol. MDMs were left untreated or stimulated with CL075 (Invivogen) at 2 μg/mL for 60 min, with four technical replicates per condition. Fixation, immunostaining with anti-human IRF5 mAb (Abcam, #10T1) and anti-human p65 XP mAb (Cell Signaling Technology, #8242, Danvers, MA, USA), and ScanˆR high-throughput imaging (Olympus Europa SE & Co., Hamburg, Germany) were done, as previously described in detail [13]. Quantification of IRF5 nuclear translocation was done with ScanˆR analysis software (v2.8.1) and calculated as a percentage of the positively stained nuclei multiplied by the mean fluorescence-intensity value (MFI) of the positively stained nuclei. Silencing efficiency of the TIRAP gene for each donor (*n* = 6) was examined by RT-qPCR, using the parallel 24-well plates.

### *2.10. Bacteria and Infection Experiments*

Anonymized clinical isolates of GBS, *S. aureus*, and *E. coli* were from a diagnostic collection by the Department of Medical Microbiology, St. Olavs Hospital, Trondheim, Norway. For infection experiments, blood-agar colonies were picked and grown in Todd-Hewitt Broth (GBS) or Tryptic Soy Broth (*E. coli* and *S. aureus*), with vigorous shaking at 37 ◦C overnight. The bacteria cultures were diluted to the desired density (CFU/mL), based on OD600 measurements and calculations, as previously described in detail [26]. Cultures of MDMs in 24-well plates, with *TIRAP* siRNA or control siRNA pre-treatment, were incubated with bacteria at 37 ◦C for 60 min, before the killing of all extracellular bacteria with 100 μg/mL gentamicin. The MDM cultures were incubated further for a total challenge time of four hours. Cell lysis and RNA purification was done with the RNeasy 96 Plus kit (QIAGEN), followed by cDNA synthesis with Maxima cDNA synthesis kit (Thermo Fisher Scientific), and qPCR analysis of the cytokine expression.

### *2.11. Statistical Analysis*

Data that were assumed to follow a log-normal distribution was log-transformed prior to statistical analysis. RT-qPCR was log-transformed and analyzed by Repeated Measurements Analysis of Variance (RM-ANOVA), or a mixed model if there was missing data, followed by Holm-Šídák's multiple comparisons post-test. ScanˆR data were logtransformed and analyzed with a paired *t*-test (two-sided). ELISA data was analyzed using a Wilcoxon matched-pairs signed-rank test. All graphs and analyses were generated with GraphPad Prism v9.1.2 (Dotmatics, Bishops Stortford, UK).
