Fig 1: SUMOylation regulates rhTRIM5α-mediated HIV-1 restriction. A. HeLa cells were transfected or not with rhTRIM5α and with control siRNA or siRNA targeting SUMO1, Ubc9 or PIAS1, 48 h before being challenged by increasing doses of HIV-1/GFP. The proportion of GFP-positive cells was measured by flow cytometry 48 h post-transduction. The graph shows means of duplicate values +/- SD, representative of three independent experiments. *p < 0.001 using the Huber/White/sandwich variance-covariance robust estimator (linear regression coefficients R2 are indicated). The expression levels of HA-rhTRIM5α were evaluated by western-blot (top right) using anti-HA antibodies (3 F10, Roche). The efficiency of RNA silencing was estimated by quantitative RT-PCR analysis and western-blot using anti-SUMO1 (Santa-Cruz Biotechnology sc-9060), anti-Ubc9 (Abgent, AM1261a) and anti-PIAS1 (Abcam, ab32219) antibodies (bottom right). B. Wild-type or rhTRIM5α-expressing HeLa cells were transfected with His-SUMO1 before transduction with increasing doses of HIV-1/GFP. The proportion of GFP-positive cells was measured by flow cytometry 48 h post-transduction. The graph shows means of duplicate values +/- SD, representative of three independent experiments. *p < 0.001 using the Huber/White/sandwich variance-covariance robust estimator. The expression levels of HA-rhTRIM5α were evaluated by western-blot (top right). Cell extracts from HeLa untransfected or transfected with SUMO1 were also analyzed by western-blot for SUMO1 expression (bottom right). C. hTRIM5α interacts with SUMO1 in the cytoplasm and nuclei of HeLa cells. Paraformaldehyde-fixed cells were incubated with primary rabbit anti-TRIM5α [13] and mouse anti-SUMO1 (clone 21C7, DSHB) antibodies for 1 h at 37°C. After washing slides were incubated with Duolink PLA probes (Olink Biosciences) for 1 h at 37°C. Ligation of the connector oligonucleotides, rolling-circle amplification and detection of the amplified DNA products were done with Duolink Detection Reagents Red according to the manufacturer’s instructions. Nuclei were labelled with Hoechst. Images were acquired using an LSM510 inverted microscope with a 63× objective. Scale bar indicates 5 μm.
Fig 2: PIAS1 SIRT6 co-immunoprecipitation.HEK293 cell extracts were prepared and immunoprecipitated with an antibody to PIAS1 (lane 1 αPIAS1 IP) or an irrelevant antibody (lane 2 Control IP) followed by Western blotting with anti-PIAS1 antibody. The PIAS1 band was seen to migrate just above the 62 kDa marker. HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 (lane 3) or the H133W mutant (lane 4) and immunoprecipitated with anti-Flag followed by Western blotting with anti-PIAS1 (α-Flag IP). In a separate experiment HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecipitated with an anti-Flag antibody and analysed by Western blotting with anti-PIAS1 antibody (lanes 5–8). Lane 5 represents the total cell extract used in the immunoprecipitation (input). Lane 6 represents the anti-Flag immunoprecipitation eluted from the beads with Flag peptide (α-Flag IP). Lanes 7 represents the non-immunoprecipitated “flow through” material from the cell extract (FT) and lanes 8 represents the immunoprecipitated material eluted from the beads with SDS-PAGE sample buffer (α-Flag IP).
Fig 3: HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecipitated with a variety of antibodies and analysed by Coomassie staining the SDS-PAGE (lanes 1,2) or by Western blotting with anti-SIRT6 (lanes 3–11).Lanes 2 shows a stained SDS-PAGE of a HEK293 cell extract immunoprecipitated with anti-Flag (α-Flag IP) and lane 1 shows a control where the extract was omitted (no extract control). The three bands seen in lane 2 (a,b,c) were excised and sequenced by ms (Table 2). Lanes 3–11 are all anti-SIRT6 Western blots. Lane 3 shows a control immunoprecipitation where the anti-Flag antibody was omitted from the IP (no IP antibody control). Lane 4 shows the anti-Flag immunoprecipitate and lane 5 shows a sample of the total cell extract without immunoprecipitation. Lane 6 shows a sample (150 ng) of purified recombinant SIRT6 [17]. In a separate experiment and at higher exposure of the Western blot, lane 7 shows an anti-Flag IP, lane 8 shows an anti-SMARCA5 IP, lane 9 shows an anti-PIAS1 IP, lane 10 shows an anti-MYBBP1A IP and lane 11 shows an anti-SMARCA5 IP from H133W transfected cells. The figure is a composite of more than one experiment but represents data from three repeated experiments.
Fig 4: Human and rhesus macaque TRIM5α are SUMO substrates. A. In vitro translated human or rhesus macaque TRIM5α proteins were incubated with purified recombinant E1 (SAE1 and SAE2 subunits, 370 nM), E2 (Ubc9, 630 nM) enzymes, recombinant SUMO1 or SUMO2 (7 μM), and increasing doses of GST-PIAS1 (0, 0.3 or 1.5 μM) or GST alone (0.3 or 1.5 μM) as control. Reaction products were analyzed by 10% SDS-PAGE. B. HeLa cells expressing HA-tagged hTRIM5α or rhTRIM5α were transfected with Ubc9 and 6xHis-SUMO1, when indicated (+). Cell lysates were directly analyzed by SDS-PAGE and immunoblotted with anti-HA antibodies. Alternatively, cell extracts were subjected to an immobilized-metal affinity chromatography (IMAC) on nickel-agarose beads under strongly denaturing conditions and 6xHis-SUMO conjugated proteins were analyzed by SDS-PAGE and immunoblotted with anti-HA antibodies. The position of unmodified TRIM5α is indicated by an asterisk whereas black arrows indicate the position of SUMOylated forms.
Fig 5: Small ubiquitin‐like modifier E3 ligase PIAS1 sumoylates and stabilizes leukemogenic kinase FIP1L1‐PDGFRA. (a) FIP1L1‐PDFRA is sumoylated by PIAS1. HEK293 cells were transfected with a combination of pCI‐6xMyc‐PIAS1, pFLAG‐FIP1L1‐PDGFRA‐FL, and pCGT‐SUMO‐1. The total amount of transfected vectors was 6 μg, with 2 μg each vector used and empty vector used as a mock. FLAG‐FIP1L1‐PDGFRA was detected by anti‐PDGFRA antibody and Myc‐PIAS1 was detected by anti‐Myc antibody. FIP1L1‐PDGFRA was immunoprecipitated with anti‐FLAG M2 antibody and subsequently analyzed by immunoblotting. Sumoylation of FIP1L1‐PDGFRA was detected by anti‐T7 antibody. (b) Knockdown of PIAS1 by siRNA attenuated sumoylation of FIP1L1‐PDGFRA. HEK293 cells were transfected with pFLAG‐FIP1L1‐PDGFRA‐FL and/or pCGT‐SUMO1 and/or human PIAS1‐specific siRNA. Decreased expression of endogenous PIAS1 by siRNA was confirmed by anti‐PIAS1 antibody. Decreased expression of PIAS1 was accompanied by attenuation of sumoylation of FIP1L1‐PDGFRA (lanes 3 and 4). (c) Knockdown of PIAS1 resulted in a decrease of FIP1L1‐PDGFRA. BAF‐FIP1L1‐PDGFRA‐FL cells were transfected with two different murine PIAS1‐specific siRNAs or a negative control. HEK293‐derived cells expressing FIP1L1‐PDGFRA were transfected with two different human PIAS1‐specific siRNAs or a negative control. After 2 days, the expression levels of PIAS1 and FIP1L1‐PDGFRA were analyzed by immunoblotting with anti‐PDGFRA antibody and anti‐PIAS1 antibody.
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