Fig. 1: HCMV infection induces protein citrullination.

a Protein lysates from HFFs infected with HCMV strain Merlin (HCMV) (MOI 1 PFU/cell) at 48 and 96 h post infection (hpi) or from uninfected HFFs (mock) were exposed to an Rh–PG citrulline-specific probe (left panel) and subjected to gel electrophoresis to detect citrullinated proteins. Equal loading was assessed by Coomassie blue staining (right panel). One representative gel of three independent experiments is shown. b mRNA expression levels of PADI isoforms by RT-qPCR of HCMV-infected (24 hpi) vs. uninfected (mock) HFFs were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase GAPDH and expressed as mean fold change ± SEM over mock-infected cells (n = 3 independent determinations; PADI2, PADI4, and PADI6 P < 0.001, two-way ANOVA followed by Bonferroni’s post test). c PADI2 and PADI4 mRNA levels in HCMV-infected HFFs at the indicated time points (hpi) were normalized to GAPDH mRNA and expressed as mean fold change ± SEM over mock-infected cells (n = 3 independent determinations; PADI2: mock vs. 8 hpi P < 0.001, mock vs. 16 hpi P < 0.001, mock vs. 24 hpi P < 0.001, mock vs. 32 hpi P < 0.001; PADI4: mock vs. 8 hpi P = 0.00375, mock vs. 16 hpi P < 0.001, mock vs. 24 hpi P < 0.001, mock vs. 32 hpi P = 0.04, one-way ANOVA followed by Bonferroni’s post tests). d Western blot analysis of protein lysates from uninfected (mock) or infected HFFs using antibodies against PAD2, PAD4, UL44, or α-tubulin (α-TUB). One representative blot and densitometric analysis shown of three independent experiments. Values are expressed as fold change in PAD2 and PAD4 expression normalized to α-tubulin. e Western blot analysis of protein lysates from uninfected (mock) or infected HFFs with HCMV wild-type (HCMV), or UV-inactivated HCMV (UV) (left panel), treated with 150 μg/ml CHX (left panel) at 24 hpi, or with 250 µM PFA (right panel) at 72 hpi or left untreated. Analysis was performed using antibodies against PAD2, IEA (recognizing IE1-72- and IE2-86 kDa), pp28, or ACTIN. One representative blot of three independent experiments is shown. f Western blot analysis of protein lysates from uninfected (mock), infected HFFs for 48 h with AdVIE1, AdVIE2, or AdVLacZ (MOI = 10) using antibodies against IEA (recognizing IE1-72- and IE2-86 kDa), PAD2, or ACTIN. One representative blot of three independent experiments is shown. g Western blot analysis of protein lysates from HFFs infected with wild-type AD169 HCMV (AD169) or AD169ΔIE1 (MOI 1 PFU/cell), the latter complemented with AdVLacZ or AdVIE1 (MOI 10 PFU/cell), at 24 hpi or from uninfected HFFs (mock) using antibodies against IEA (recognizing IE1-72- and IE2-86 kDa), PAD2, or ACTIN. One representative blot of three independent experiments is shown. Data are shown as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.