rfp reporter  (Addgene inc)

Journal: bioRxiv

Article Title: A proteome-wide, MS-based screen identifies SUMOylation of host RNA splicing factors induced by HIV-1 infection

doi: 10.1101/2025.03.26.645526

Figure Lengend Snippet: (A) Western blot showing expression of free and conjugated forms of 3xFLAG-SUMO1 Q92R and 3xFLAG-SUMO2 Q88R in HeLa cells. Expression of endogenous wild-type SUMO is shown for comparison. “-” = Untransfected Control. (B) SUMO-modified RanGAP1 is detected following immunoprecipitation with a FLAG or HIS antibody in cells expressing 3xFLAG-SUMO1 Q92R or HIS 6 -SUMO1 Q92R , respectively. Whole cell lysate (WCL) was used as input for IPs. Non-immune IgG = IP control. (C) Expression of exogenous SUMO does not interfere with HIV-1 infection. Cell lines were infected with equal volumes of viral supernatants containing VSV-G pseudotyped HIV-1 pNL4-3 deltaENV-EGFP reporter virus. %GFP+ cells and GFP mean fluorescence intensity (MFI) was assessed by flow cytometry at 24 hpi. “-” = Untransfected Control.

Article Snippet: Cells were co-transfected with 15 µg of the pNL4-3 deltaENV-EGFP reporter (Centre for AIDS reagents #100616) and 5 µg of the pCMV-VSV-G vector (Addgene #8454) using Lipofectamine 3000 (Invitrogen #L3000075, per manufacturer’s protocol) in serum-free DMEM.

Techniques: Western Blot, Expressing, Comparison, Control, Modification, Immunoprecipitation, Infection, Virus, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: A proteome-wide, MS-based screen identifies SUMOylation of host RNA splicing factors induced by HIV-1 infection

doi: 10.1101/2025.03.26.645526

Figure Lengend Snippet: (A) Western blot showing the level of SUMO1-modified and unmodified RanGAP1 in HeLa cells treated with the SUMOylation inhibitor TAK-981 versus the DMSO control (“-”). (B) Western blot showing the level of SUMO1-conjugated A2B1 and A3 in HIV-1-infected cells treated with or without TAK-981. Uninfected, untreated cells were used as a control. Crude lysate from the corresponding conditions was used as input for IPs (5% of total protein loaded in IPs). For both A and B, TAK-981 was used at a concentration of 12 µM. DMSO = untreated control. (C) Western blot analysis of SUMO1-modified A2B1 and A3 in cells infected with Env mutant HIV-1 (pNL4-3 deltaENV-EGFP) lacking the VSV-G envelope (“-VSV-G HIV”) versus the uninfected control. Cells were harvested at 48 hpi. (D) Western blot showing the level of SUMO-modified A2B1 or A3 in HIV-1-infected HeLa cells versus uninfected controls at 6, 12, 24, and 48 hpi. (E, F) Western blot analysis of SUMO1-modified A2B1 and A3 in HIV-1-infected Jurkat cells (E) or K562 cells (F) versus uninfected controls at 48 hpi. (G) Western blot analysis of SUMO1-modified A2B1 and A3 in MMLV-infected HeLa cells versus uninfected controls. Cells were infected with VSV-G pseudotyped MMLV (pNCA-GFP) and harvested at 48 hpi for lysate preparation. Crude lysate input for each condition is shown for comparison. For all, SUMO1-conjugated proteins were immunoprecipitated using anti-SUMO1 antibodies and samples were analyzed by western blot for A2B1 and A3. Arrows indicate SUMO1-modified proteins. Stars indicate unmodified proteins. For C-G, non-immune IgG = IP control and SUMO1-modified RanGAP1 = loading control for IPs.

Article Snippet: Cells were co-transfected with 15 µg of the pNL4-3 deltaENV-EGFP reporter (Centre for AIDS reagents #100616) and 5 µg of the pCMV-VSV-G vector (Addgene #8454) using Lipofectamine 3000 (Invitrogen #L3000075, per manufacturer’s protocol) in serum-free DMEM.

Techniques: Western Blot, Modification, Control, Infection, Concentration Assay, Mutagenesis, Comparison, Immunoprecipitation