dependent p14ARF methylation in response to DNA damage.Figure 5HeLa cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and two PRMT1-specific siRNAs) and irradiated at 150 J/cm2 UVC or not irradiated. After 24 hours, the cellular distribution of endogenous p14ARF was determined by immunofluorescence (IF) staining using α-p14ARF antibody (red, endogenous p14ARF) and in the merge additionally DAPI (blue, nuclei/DNA). Representative IF results are shown for two conditions (siControl_4 and siPRMT1_1) in (A), with asterisks indicating exclusively nucleolar and arrowheads indicating not-exclusively nucleolar but additionally or predominantly nucleo-/cytoplasmic p14ARF localization. Scale bars: 15 μm. The subcellular distribution of p14ARF, as determined by IF, was quantified by cell counting (percentage of cells) for all conditions (B). PRMT1 depletion and p14ARF protein levels were analyzed by immunoblotting using the indicated antibodies (C). The p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blot, with the siControl_4 condition set to 1. D: GST-tagged p14ARF and PRMT1 proteins purified from bacteria were subjected to an in vitro-methyltransferase (MT) assay in the presence of SAM. Reactions were separated by SDS-PAGE and analyzed by immunoblotting using α-me-p14ARF and α-p14ARF antibodies. E: U2OS cells were transfected with Flag-tagged wild type (wt) or mutant (RK) p14ARF-containing plasmids. Methylation and overexpression of p14ARF were analyzed by immunoblotting using α-me-p14ARF, α-p14ARF and α-β-TUBULIN (loading control) antibodies. The me-p14ARF antibody predominantly recognizes the p14ARF protein on the immunoblot, but weakly also other protein bands with higher molecular weights. F: HeLa cells were irradiated at 150 J/cm2 UVC. After 0, 8, 16, and 24 hours, methylation of endogenous p14ARF was analyzed by immunoblotting using α-me-p14ARF, α-p14ARF and α-β-TUBULIN (loading control) antibodies. The methylated p14ARF and the p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blots, with the not irradiated condition (0 hour) set to 1. G: HeLa cells either CRISPR/Cas9 control (CTR) or PRMT1-deleted (KO_1) were irradiated at 150 J/cm2 UVC. After 0, 2, and 4 hours, cell lysates were analyzed by immunoblotting using the indicated antibodies. Depicted staining results derive from the same blot as well as exposure times with white lines (between CTR and KO_1) indicating where tracks were cut. The percentage of PARP cleavage was densitometrically quantified and is indicated below the blot. H: HeLa cells were irradiated at 150 J/cm2 UVC. After 0, 2, 4, and 6 hours, immunoprecipitation (IP) of endogenous p14ARF was performed from cell lysates using α-p14ARF antibody or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies. Short and long exposure times are indicated. The p14ARF and the methylated p14ARF signals were densitometrically quantified and normalized to the respective β-TUBULIN signal, as specified by the numbers below the blots, with the not irradiated condition (0 hour) set to 1. The percentage of PARP cleavage was also densitometrically quantified and is indicated below the blot.