Journal: STAR Protocols

Article Title: Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis

doi: 10.1016/j.xpro.2020.100089

Figure Lengend Snippet:

Article Snippet: Transfer proteins to a membrane using the eBlot L1 Blotting System (GenScript).

Techniques: Recombinant, Modification, Protease Inhibitor, Clear Native PAGE, Membrane, Bicinchoninic Acid Protein Assay

Journal: STAR Protocols

Article Title: Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis

doi: 10.1016/j.xpro.2020.100089

Figure Lengend Snippet: eBlot L1 Transfer Buffer (GenScript) as Explained by the Supplier

Article Snippet: Transfer proteins to a membrane using the eBlot L1 Blotting System (GenScript).

Techniques: Concentration Assay, Membrane

Journal: STAR Protocols

Article Title: Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis

doi: 10.1016/j.xpro.2020.100089

Figure Lengend Snippet: Other Solutions

Article Snippet: Transfer proteins to a membrane using the eBlot L1 Blotting System (GenScript).

Techniques: Clear Native PAGE, Membrane

Journal: Journal of Virology

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

doi: 10.1128/JVI.02828-13

Figure Lengend Snippet: APOL1 inhibits HIV-1production and infectivity. (A) 293T cells were seeded in 6-well plates and transfected with the indicated combination HIV-1 proviral construct DNA (1 μg), A3G (1 μg), and APOL1 (1 μg) expression vectors. Twenty hours after transfection, the whole-cell lysates (50 μg/lane) and pelleted viruses were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting for HIV-1 protein expression. (B) APOL1 inhibits intracellular accumulation of HIV-1 Vpu. 293T cells were cotransfected with the indicated HIV-1 proviral DNA and APOL1 or pcDNA3.1 vectors. The whole-cell lysates were analyzed by immunoblotting for the expression of Vpu, APOL1, and actin. (C) APOL1 depletes cellular HIV-1 Gag and stimulates accumulation of lysosomes. 293T cells were transfected with 1 μg of HIV-1 89.6 and 0.5 μg of pcDNA3.1 (control) or 0.5 μg of APOL1-myc vector (APOL1). Five hours later, the cells were trypsinized and a fraction of transfected cells was transferred into 2-well chamber dishes. Twenty hours after transfection, the cells were incubated for 30 min with LysoTracker red (100 nM), followed by fixation, permeabilization, and staining with rabbit anti-p17 Gag and goat anti-rabbit Alexa 488. For APOL1 labeling, the permeabilized cells were stained with mouse anti-myc and goat anti-mouse Alexa 647 antibodies. The cells were mounted with SlowFade with DAPI to stain nuclei (blue) and subjected to confocal microscopy. Number of lysosomes/cell (red fluorescent puncta) were determined and are presented as means ± SDs from 10 control cells and from 10 cells expressing APOL1 (pink). (D) APOL1 reduces HIV-1 infectivity. 293T cells were transfected with NL4-3 or JR-CSF alone or in combination with A3G and APOL1 (1 μg of DNA each). Cell supernatants containing released viruses were filtered, and equal amounts of HIV-1 (normalized by p24 ELISA) were added to TZM-bl indicator cells in triplicate. The relative infectivity of viruses was determined by a luciferase assay. Infectivity of the virus produced by cells transfected with HIV-1 proviral DNA only was set as 1.0 (control). Error bars indicate SDs.

Article Snippet: After 24 h, the cells were tested by real-time PCR for the expression levels of APOL1 transcripts using APOL1-specific primers (OriGene) and by immunoblotting for the expression of APOL1 protein.

Techniques: Infection, Transfection, Construct, Expressing, SDS Page, Western Blot, Plasmid Preparation, Incubation, Staining, Labeling, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Luciferase, Produced

Journal: Journal of Virology

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

doi: 10.1128/JVI.02828-13

Figure Lengend Snippet: APOL1 promotes nuclear localization of TFEB and stimulates expression of lysosomal genes. (A) 293T cells were transfected with TFEB-GFP (1 μg) and APOL1-DsRed (APOL1) (1 μg) or pcDNA3 (control) (1 μg), and 5 h after transfection, a fraction of transfected cells was transferred into 2-well chamber dishes. Twenty-four hours after transfection, the cells were fixed, mounted in SlowFade with DAPI to stain nuclei (blue), and subjected to confocal microscopy. (B) Quantification of the subcellular distribution of TFEB-GFP in the absence (control) and presence of APOL1-DsRed (APOL1). The percentages of cells with cytoplasmic (C) or nuclear (N) localization of TFEB-GFP or both (C + N) is shown. ***, P < 0.001 for comparison of differences between nuclear TFEB-GFP ([N] + [C + N]) in cells transfected with TFEB-GFP (bar 1) and TFEB-GFP with APOL1-DsRed (bar 3). Nuclear localization of TFEB in the presence of 100 μM chloroquine (CLQ) was used as a positive control (36). (C) Real-time PCR analysis of TFEB target gene expression. 293T cells were transfected in triplicate with indicated increasing amounts of APOL1, total RNA was isolated, and synthesized cDNA was subjected to qPCR to detect expression of endogenous LAMP1, LAMP2, CD63, and TFEB RNAs (64). Gene expression was normalized to GAPDH mRNA expression. Error bars indicate SDs.

Article Snippet: After 24 h, the cells were tested by real-time PCR for the expression levels of APOL1 transcripts using APOL1-specific primers (OriGene) and by immunoblotting for the expression of APOL1 protein.

Techniques: Expressing, Transfection, Staining, Confocal Microscopy, Positive Control, Real-time Polymerase Chain Reaction, Isolation, Synthesized

Journal: Journal of Virology

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

doi: 10.1128/JVI.02828-13

Figure Lengend Snippet: APOL1 inhibits HIV-1 Gag RNA expression. (A) 293T cells were transfected in triplicate with 1GA NL4-3 (22) in the absence or presence of the indicated amounts of APOL1 DNA expression vector. After 24 h, the total cellular RNA was extracted and cDNA synthesized. The expression of HIV-1 Gag RNA was analyzed by real-time qPCR analysis using HIV-1 p24 primer sets (24). The HIV-1 Gag RNA levels were normalized against GAPDH mRNA. (B) 293T cells were transfected with HIV-1 proviral construct DNA (1 μg), pEGFP-N1 (1 μg), and the indicated amounts of APOL1 expression vector. Twenty hours after transfection, the whole-cell lysates (40 μg per lane) and pelleted viruses were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting for the expression of Gag, APOL1, enhanced GFP (EGFP), and actin. Intensities of Gag and EGFP signals were obtained by densitometry. The expression of total Gag and EGFP was normalized against the expression of actin and presented as Gag/actin and EGFP/actin ratios. Error bars indicate SDs.

Article Snippet: After 24 h, the cells were tested by real-time PCR for the expression levels of APOL1 transcripts using APOL1-specific primers (OriGene) and by immunoblotting for the expression of APOL1 protein.

Techniques: RNA Expression, Transfection, Expressing, Plasmid Preparation, Synthesized, Construct, SDS Page, Western Blot

Journal: Journal of Virology

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

doi: 10.1128/JVI.02828-13

Figure Lengend Snippet: APOL1 stimulates endocytosis and increases lysosome accumulation. (A) 293T cells in 6-well plates were transfected with pcDNA3.1 (control) (1 μg) or APOL1 expression vector (APOL1) (1 μg). Transfection efficiency in 293T cells was determined by transfecting the APOL1-GFP construct (1 μg) and counting GFP-positive cells by fluorescence microscopy. We observed more than 90% GFP-positive cells (data not shown). Sixteen hours after transfection, the cells were incubated with Oregon Green 488 dextran (250 μg/ml) for 4 h at 37°C, followed by 2 h in dextran-free complete medium. After a washing with PBS, live cells were imaged by fluorescence microscopy. To quantitate the uptake of fluorescently labeled dextran by cells, the cells were trypsinized, washed, and analyzed by FACS. A representative experiment is shown. (B) 293T cells transfected as for panel A were incubated with DQ-Green BSA (250 μg/ml) for 2 h at 37°C followed by 1 h in DQ-Green BSA-free complete medium. After a washing, the cells were incubated for 30 min in medium containing LysoTracker red (100 nM) and then washed, and images were obtained by fluorescence microscopy. (C) The uptake of DQ-Green BSA into lysosomes and the mass of LysoTracker red-positive lysosomes were quantitated by FACS as described for panel B, with the exception that DQ-Green BSA was used at 25 μg/ml. (D) 293T cells were transfected with increasing amounts of APOL1 vector (transfected DNA was balanced with pcDNA3.1), and after 18 h, the cell lysates were analyzed by immunoblotting for the expression of LAMP1, APOL1, and actin. Intensities of protein signals were obtained from densitometric scanning. The expression of LAMP1 was normalized against the expression of actin and presented as the LAMP1/actin ratio. In cells transfected with pcDNA3.1 only, the LAMP1/actin ratio was used as a reference.

Article Snippet: After 24 h, the cells were tested by real-time PCR for the expression levels of APOL1 transcripts using APOL1-specific primers (OriGene) and by immunoblotting for the expression of APOL1 protein.

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Fluorescence, Microscopy, Incubation, Labeling, Western Blot

Journal: Journal of Virology

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

doi: 10.1128/JVI.02828-13

Figure Lengend Snippet: APOL1 reduces stability of HIV-1 Gag. (A) 293T cells were transfected with 1GA NL4-3 and APOL1 expression vector at a ratio of 2:1. Control cells were transfected with 1GA NL4-3 and pcDNA3. Eighteen hours after transfection, cells were labeled for 30 min with [35S]methionine-cysteine (25). Cell lysates were collected at the indicated chase times, and HIV-1 Gag was immunoprecipitated using rabbit anti-p17 antibody and protein A/G-Plus. Complexes were resolved by 10% SDS-PAGE and gels exposed to X-ray film. (B) Intensities of the Gag p55 and p24 bands were quantified using Bio-Rad ChemiDoc Imager and Quantity One software. Total Gag half-life values were normalized to chase time zero values. Gag half-life values in cells cotransfected with APOL1 or pcDNA3 were calculated using regression analysis.

Article Snippet: After 24 h, the cells were tested by real-time PCR for the expression levels of APOL1 transcripts using APOL1-specific primers (OriGene) and by immunoblotting for the expression of APOL1 protein.

Techniques: Transfection, Expressing, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Software

Journal: Journal of Virology

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

doi: 10.1128/JVI.02828-13

Figure Lengend Snippet: APOL1 depletes intracellular Vif by lysosomal degradation and stimulating its secretion in microvesicles. (A) 293T cells were transfected with Vif-expressing pNL-A1 vector alone or in combination with APOL1 expression vector. Five hours after transfection, the cells were treated with E64d (10 μg/ml) and pepstatin A (10 μg/ml). After 24 h, whole-cell lysates and pelleted microvesicles were separated by 10% SDS-PAGE and analyzed by Western blotting for the expression of Vif, APOL1, and microvesicle markers Alix and CD81. Secretion of microvesicles was inhibited by incubation of transfected cells with the calcium chelator BAPTA-AM (10 μM) for 16 h. (B) Depletion of Rab7A with siRNA inhibited lysosomal degradation of Vif and potentiated APOL1-mediated release of Vif in microvesicles. 293T cells were transfected twice with Rab7A siRNA, followed by transfection with pNL-A1 alone or with APOL1 vector. After 24 h, whole-cell lysates and pelleted microvesicles were separated by 10% SDS-PAGE and analyzed by Western blotting for the expression of Vif, APOL1, and Rab7 and microvesicle markers Alix and Tsg101. Co, control.

Article Snippet: After 24 h, the cells were tested by real-time PCR for the expression levels of APOL1 transcripts using APOL1-specific primers (OriGene) and by immunoblotting for the expression of APOL1 protein.

Techniques: Transfection, Expressing, Plasmid Preparation, SDS Page, Western Blot, Incubation

Journal: Journal of Virology

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

doi: 10.1128/JVI.02828-13

Figure Lengend Snippet: Inhibition of endocytosis by depletion of Eps15, Stx7, Rab7, or VAMP7 restores virus production in cells expressing APOL1. 293T cells were transfected on day 1 and day 2 with control siRNA (Co) or siRNAs that specifically target Eps15 (A), Stx7 (B), Rab7 (C), VAMP7 (D), or Stx17 (E). Twenty-four hours later, the cells were transfected with HIV-1 89.6 proviral DNA in the presence or absence of APOL1 vector. After an additional 24 h, pelleted virus from culture supernatants and corresponding cell lysates were collected and analyzed by immunoblotting for the expression of Gag, APOL1, and proteins targeted by siRNA. (E) Depletion of the autophagosomal marker Stx17 did not restore virus production by cells overexpressing APOL1.

Article Snippet: After 24 h, the cells were tested by real-time PCR for the expression levels of APOL1 transcripts using APOL1-specific primers (OriGene) and by immunoblotting for the expression of APOL1 protein.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Western Blot, Marker

Journal: Journal of Virology

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

doi: 10.1128/JVI.02828-13

Figure Lengend Snippet: IFN-γ-stimulated expression of APOL1 in differentiated U937 monocytes contributes to inhibition of HIV-1 production. (A) APOL1 expression is inducible by IFN-γ in differentiated human monocytes (THP-1 and U937) but not in CD4+ T cells (SupT1 and Jurkat). The cells were stimulated for 24 h with PMA (50 ng/ml) or were left untreated (control). After a washing, PMA-treated cells were cultured in media alone (PMA) or were treated for another 24 h with IFN-γ (20 ng/ml) (PMA/IFN-γ). As an additional control, 293T cells were transfected with APOL1 vector (0.5 μg). Cell lysates (50 μg/lane) were analyzed by immunoblotting for the expression of APOL1 and actin. (B) (Left side) Seven-day differentiated MDM were either left untreated or stimulated for 18 h with 2 different doses (25 or 50 ng/ml) of IFN-γ. Cell lysate proteins (50 μg/lane) were resolved by 10% SDS-PAGE, and the levels of APOL1, IRF-1, and Samhd1 were analyzed by Western blotting. Expression of transcription factor IRF-1 served as a positive control for IFN-γ treatment (45). (Right side) Seven-day differentiated MDM were either left untreated (control) or stimulated for 18 h with IFN-α or IFN-γ at 100 ng/ml. Cell lysate proteins were resolved by 4 to 12% gradient SDS-PAGE, and the levels of APOL1, IRF-1, A3A, and Samhd1 were analyzed by Western blotting. (C) U937 clones expressing control shRNA (control) or APOL1 shRNA (APOL1) were stimulated for 24 h with PMA (50 ng/ml), followed by treatment with IFN-γ (20 ng/ml) as indicated. After 24 h, the cells were rinsed and exposed to HIV-1 89.6 (50 ng of p24 per 106 cells) pseudotyped with VSV-G. After 4 h of incubation, the cells were washed and cultivated for another 48 h, followed by collection of supernatants and cell lysates. Pelleted virus and lysates were analyzed by immunoblotting for the expression of HIV-1 Gag, APOL1, and actin. Virus production was determined by the expression of pelletable p24. Protein signals were obtained from densitometric scanning. Virus released from PMA-treated U937-CoshRNA cells (control) was used as a reference (100%).

Article Snippet: After 24 h, the cells were tested by real-time PCR for the expression levels of APOL1 transcripts using APOL1-specific primers (OriGene) and by immunoblotting for the expression of APOL1 protein.

Techniques: Expressing, Inhibition, Cell Culture, Transfection, Plasmid Preparation, Western Blot, SDS Page, Positive Control, Clone Assay, shRNA, Incubation

Journal: Journal of Virology

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

doi: 10.1128/JVI.02828-13

Figure Lengend Snippet: Proposed model for APOL1-mediated inhibition of HIV-1 expression. (Left side) In the absence of APOL1, HIV-1 Gag is mainly targeted to the plasma membrane, where virus assembly takes place. Expression of HIV-1 Vif depletes cells of A3G and preserves infectivity of the virus progeny. (Right side) In the presence of APOL1, transcription of HIV-1 is partly inhibited, which leads to decreased synthesis of Gag protein. Gag protein is targeted for degradation in the lysosomal compartment, which expands due to stimulation of lysosome biogenesis induced by nuclear translocation of TFEB in cells expressing APOL1. Here, Gag is also targeted to the plasma membrane, but increased endocytosis redirects Gag to endosomes that efficiently fuse with an increased pool of lysosomes. Infectivity of produced virions is partly reduced by APOL1-dependent suppression of HIV-1 transcription and reduction of cellular Vif levels by degradation in lysosomes and secretion in microvesicles (Exo). As a result, host restriction factor A3G is not degraded but encapsidated and reduces HIV-1 virion infectivity. LYS, lysosomes; LE/MVB, late endosomes or multivesicular bodies; EE, early endosomes.

Article Snippet: After 24 h, the cells were tested by real-time PCR for the expression levels of APOL1 transcripts using APOL1-specific primers (OriGene) and by immunoblotting for the expression of APOL1 protein.

Techniques: Inhibition, Expressing, Infection, Translocation Assay, Produced