α-senp3 antibody  (Santa Cruz Biotechnology)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation *

doi: 10.1074/mcp.M116.064741

Figure Lengend Snippet: HIN domain of IFIX mediates its interaction with the 5FMC complex. A, validating IFIX co-interaction with SENP3 by reciprocal IP. B, IFIX interacts with 5FMC components LAS1L and SENP3 through its HIN domain. Forward IPs using GFP antibody were performed in cells transfected with IFIX constructs full-length (FL), IFIX Pyrin domain (PY), and IFIX HIN200 domain (HIN) in pEGFP. Inputs (1.5%), elutions (20%), and isolated IFIX constructs (IP, 20%) were blotted for LAS1L, SENP3, and GFP. C, IF microscopy in IFIX-GFP and EGFP control 293 cells showing a redistribution of 5FMC protein LAS1L in infected cells (white arrows), m.o.i.: 5, 4 hpi. Co-localization of IFIX and LAS1L is pronounced in uninfected cells (for PELP1, see supplemental Fig. S3). D, Levels of PELP1 and LAS1L are not reduced during HSV-1 infection. PELP1 and LAS1L levels were monitored by western blotting at 6hpi. ICP27 is marker for infection. M, mock. Microscopy images were taken at ×60 oil objective. Bar, 5 μm.

Article Snippet: Reagents The antibodies used for immunoaffinity purifications, Western blotting, and immunofluorescence were as follows: an in-house-generated α-green fluorescent protein (GFP) for IPs ( 25 ); α-GFP (Roche Applied Science) for Western blottings; α-PYHIN1 (Sigma-Aldrich); α-IFI16 antibodies (ab50004 and ab55328, Abcam, Cambridge, MA) used at a 1:1 mixture as described previously ( 22 ); α-ICP0 (H1A027-100, Virusys Corp., Taneytown, MD); α-ICP4 (sc-69809, Santa Cruz Biotechnology, Dallas, TX); α-ICP27 (sc-69806, Santa Cruz Biotechnology); α-ICP8 (sc-53329, Santa Cruz Biotechnology); α-tubulin (T6199, Sigma-Aldrich); α-PML (sc-9862, Santa Cruz Biotechnology); α-proteasome 20S core subunit (PW8155-0100, Enzo Life Sciences, Farmingdale, NY); α-HUWE1 (NB100-652, Novus, Littleton, CO); α-SENP3 (sc-67076, Santa Cruz Biotechnology); and α-LAS1L (ab140656) and α-PELP1 (sc-393534, Santa Cruz Biotechnology).

Techniques: Transfection, Construct, Isolation, Microscopy, Infection, Western Blot, Marker

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation *

doi: 10.1074/mcp.M116.064741

Figure Lengend Snippet: IFIX inhibits viral gene transcription. A, confirmation of IFIX is overexpressed in stable HFFs. B, relative mRNA levels of viral genes ICP27 and ICP8 determined by quantitative PCR. Cells were infected with WT HSV-1 at an m.o.i. of 10 and collected at 6 hpi. Error bars represent S.D. of three biological replicates. Significance was determined by t test. *, p ≤ 0.05; **, p ≤ 0.005. C, IFIX-GFP localizes to viral genomes when its degradation is inhibited. Cells were treated with MG132 to block the proteasome during the course of 3- or 6-h infections. ICP4 is marker for viral genomes. Size bars, 5 μm. D, doubling times of HFFs stably expressing EGFP or IFIX-GFP. E, siRNA-mediated knockdown of 5FMC components PELP1 and SENP3 impact HSV-1 progeny titers in human fibroblasts. EGFP or IFIX-GFP HFFs were transfected with the indicated siRNAs. Cells were infected with WT HSV-1 at an m.o.i. of 10 at 24 h post-transfection. N.T., non-targeted. Error bars indicate S.D. of two biological replicates in technical duplicate; significance was determined by t test. *, p ≤ 0.05; **, p ≤ 0.005. n.s., not significant. F and G, validation of knockdown efficiency of PELP1 and SENP3 by Western blotting.

Article Snippet: Reagents The antibodies used for immunoaffinity purifications, Western blotting, and immunofluorescence were as follows: an in-house-generated α-green fluorescent protein (GFP) for IPs ( 25 ); α-GFP (Roche Applied Science) for Western blottings; α-PYHIN1 (Sigma-Aldrich); α-IFI16 antibodies (ab50004 and ab55328, Abcam, Cambridge, MA) used at a 1:1 mixture as described previously ( 22 ); α-ICP0 (H1A027-100, Virusys Corp., Taneytown, MD); α-ICP4 (sc-69809, Santa Cruz Biotechnology, Dallas, TX); α-ICP27 (sc-69806, Santa Cruz Biotechnology); α-ICP8 (sc-53329, Santa Cruz Biotechnology); α-tubulin (T6199, Sigma-Aldrich); α-PML (sc-9862, Santa Cruz Biotechnology); α-proteasome 20S core subunit (PW8155-0100, Enzo Life Sciences, Farmingdale, NY); α-HUWE1 (NB100-652, Novus, Littleton, CO); α-SENP3 (sc-67076, Santa Cruz Biotechnology); and α-LAS1L (ab140656) and α-PELP1 (sc-393534, Santa Cruz Biotechnology).

Techniques: Real-time Polymerase Chain Reaction, Infection, Blocking Assay, Marker, Stable Transfection, Expressing, Transfection, Western Blot