Journal: bioRxiv

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) LANA Prevents KSHV Episomes from Degradation

doi: 10.1101/2023.08.18.553898

Figure Lengend Snippet: Lytic reactivation after depletion of LANA. (A) Immunofluorescence microscopy. iSLK-OsTIR1-mAID-LANA BAC16 cells were treated with or without 2 μM 5-Ph-IAA for 24 h, treated with or without 1 μg/ml doxycycline plus 1.5 mM sodium butyrate (± Dox/NaB) for another 24 h, and then subjected to immunofluorescence staining with anti-FLAG, LANA and K8α antibodies. Bar, 100 μm. (B) Western blotting. iSLK-OsTIR1-mAID-LANA BAC16 cells were treated with 2 μM 5-Ph-IAA for 0, 1.5, 3, 6, and 24 h, and then treated with or without 1 μg/ml doxycycline plus 1.5 mM sodium butyrate (± Dox/NaB) for 24 h. The total cell lysate was prepared and subjected to immunoblotting with indicated antibodies. (C) Real-time qPCR. Total RNA was extracted and subjected to real-time qPCR. 18s rRNA was used as an internal standard for normalization, and the 5-Ph-IAA (-) Dox/NaB (-) was set as 1.

Article Snippet: Anti-FLAG M2 mouse monoclonal and anti-FLAG rabbit polyclonal antibodies, anti-LANA rat monoclonal antibody, anti-β-actin mouse monoclonal antibody, and polyvinylidene difluoride (PVDF) membrane were purchased from Millipore-Sigma (Burlington, MA, USA).

Techniques: Immunofluorescence, Microscopy, Staining, Western Blot

Journal: STAR Protocols

Article Title: Detection of membrane-anchoring lipid modifications of proteins in cells by radioactive metabolic labeling

doi: 10.1016/j.xpro.2023.102416

Figure Lengend Snippet: Detection of palmitoylation and prenylation by radioactive metabolic labeling (A) HEK293T (lane 1) and MCF10CA1a (lanes 2 and 3 as replicates) cells were labeled overnight with H 3 -palmitic acid using the protocol described in this manuscript. Post-labeling, cells were harvested, and whole cell lysates were processed to obtain soluble protein fractions using the protocol. Soluble protein fractions were subjected to SDS-PAGE using pre-cast gradient Bolt TM 4%–12% Bis-Tris Plus gels and transferred to a PVDF membrane. Subsequently, the PVDF membrane was exposed to a phosphor screen for about 2–3 weeks and H 3 -labeled proteins were detected with the phosphor imager. Top: The autoradiograph shows the profile of palmitoylated proteins in HEK293T and MCF10CA1a cell lines. Bottom: PVDF membrane was stained with ponceau S to visualize the inputs in the lanes. (B) HEK293T cells (3 replicates) were labeled overnight with H 3 -mevalonolactone according to the protocol described in this manuscript. Post-labeling cells were processed as in (A). The autoradiograph, developed after 2 weeks exposure to phosphor screen, shows that various proteins are prenylated in HEK293T cells. (C) FLAG-tagged HRAS construct was transfected in HEK293T cells and concomitantly labeled with H 3 -palmitic acid overnight using the protocol described in this manuscript. Non-transfected (NT) control cells were also labeled for the same time. Post labeling and cell lysis, HRAS was immunoprecipitated using anti-FLAG-resin from soluble protein fractions. Immunocomplex was subjected to SDS-PAGE and transferred to a PVDF membrane. PVDF membrane was exposed to the phosphor screen for about 3 weeks before detection of the label with the phosphor imager (details described in the protocol). Top panel: The autoradiograph shows specific detection of palmitoylated FLAG-HRAS, but not in non-transfected control (NT). Refer to Liang et al. wherein palmitoylation-deficient HRAS control is included under similar experimental settings. Portions of the immunoprecipitated fractions (middle panel) and inputs (bottom panel) were probed with anti-FLAG antibody to detect the corresponding FLAG-HRAS compared to NT control. (D) FLAG-tagged FBXL2 and FBXL2 (C420S) constructs were transfected in HEK293T cells and concomitantly labeled with H 3 -mevalonolactone using the protocol described in this manuscript. FLAG-FBXL2 (C420S), a prenylation-deficient mutant, serves as a control. Post-labeling and cell lysis, anti-FLAG immunoprecipitations were carried out using anti-FLAG resin and processed as in (B) and the label was detected after 2-week exposure with the phosphor imager. Top panel: The autoradiograph shows the prenylation of FLAG-FBXL2, but not FLAG-FBXL2 (C420S). The same PVDF membrane was probed with anti-FLAG antibody (middle panel) to detect corresponding FLAG-FBXL2 and prenylation deficient FLAG-FBXL2 (C420S). Bottom panel shows inputs probed with anti-FLAG antibody. ∗ Non-specific bands (Refer to Liang et al. wherein non-transfected control is included under similar experimental settings).

Article Snippet: Mouse monoclonal anti-FLAG-M2 affinity gel , Millipore Sigma , Cat#A2220.

Techniques: Labeling, SDS Page, Autoradiography, Staining, Construct, Transfection, Lysis, Immunoprecipitation, Mutagenesis

Journal: STAR Protocols

Article Title: Detection of membrane-anchoring lipid modifications of proteins in cells by radioactive metabolic labeling

doi: 10.1016/j.xpro.2023.102416

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-FLAG-M2 affinity gel , Millipore Sigma , Cat#A2220.

Techniques: Recombinant, Protease Inhibitor, Software, Blocking Assay

Journal: Viruses

Article Title: The RL13 Temperance Factor Represses Replication of the Highly Cell Culture-Adapted Towne Strain of Human Cytomegalovirus

doi: 10.3390/v15041023

Figure Lengend Snippet: Detection and localization of RL13-FLAG expressed by virus TL12-RL13 WT-FLAG . ( A ) MRC-5 cultures were infected with a p0 stock of TL12-RL13 WT-FLAG or with p10 stocks of TL12-RL13 WT-FLAG–1 or TL12-RL13 WT-FLAG–2 . At day eight post-infection, cultures were stained for FLAG, and images of the FLAG (red) and GFP (green) signal were acquired. The white arrow indicates RL13 WT-FLAG accumulation into juxtanuclear structures corresponding to the VAC. Original magnification: 100×. ( B ) MRC-5 cultures were infected with p0 stocks of TL12-RL13 WT-FLAG , TL12-RL13 E208K-FLAG , or TL12-RL13 FS-FLAG . Cell lysates prepared at day five post-infection were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed using α-FLAG, α-IE1/2, or α-β-actin monoclonal antibodies. The positions of protein molecular weight standards are indicated.

Article Snippet: Infected MRC-5 cultures in chamber slides were fixed in 1% formaldehyde for 30 min. at room temperature, permeabilized with 0.5% Triton X-100 for 20 min. on ice, blocked with 20% FBS in PBS (blocking buffer) for 30 min. at room temperature, then incubated with mouse monoclonal α-FLAG antibody M2 (1:500, Millipore-Sigma, St. Louis, MI, USA) for one hour at room temperature.

Techniques: Infection, Staining, SDS Page, Molecular Weight

Journal: Viruses

Article Title: The RL13 Temperance Factor Represses Replication of the Highly Cell Culture-Adapted Towne Strain of Human Cytomegalovirus

doi: 10.3390/v15041023

Figure Lengend Snippet: RL13-FLAG localizes to VAC and is mis-localized by the E208K substitution. MRC-5 monolayers were infected with p0 stocks of TL12-RL13 WT-FLAG ( A ) or TL12-RL13 E208K-FLAG ( B ). After four or 11 days, the cultures were imaged for GFP (green), stained for RL13 with an α-FLAG antibody (red), or stained for pp28 with an α-pp28 antibody (blue). Original magnification: 200×.

Article Snippet: Infected MRC-5 cultures in chamber slides were fixed in 1% formaldehyde for 30 min. at room temperature, permeabilized with 0.5% Triton X-100 for 20 min. on ice, blocked with 20% FBS in PBS (blocking buffer) for 30 min. at room temperature, then incubated with mouse monoclonal α-FLAG antibody M2 (1:500, Millipore-Sigma, St. Louis, MI, USA) for one hour at room temperature.

Techniques: Infection, Staining