Journal: EMBO Reports

Article Title: GABARAP proteins regulate the packaging of HIV-1 genomic RNA into virions

doi: 10.1038/s44319-025-00607-1

Figure Lengend Snippet: ( A ) HeLa cells were transfected with WT NL4-3 HIV-1 proviral DNA or non-transfected as a control. Supernatants were collected 48 h later, filtered and ultracentrifuged on a 20% sucrose cushion. Virions-enriched preparations were subsequently lysed and analyzed by LC-MS. ATG proteins (41) identified by LC-MS analysis in preparation from mock and NL4-3 HIV-1 transfected HeLa cells, are shown in a scatter plot of fold change (HIV-1 vs mock) and −log10( p value) (the hatched line represents the p value = 0.05). Blue dots indicate proteins controlling the ESCRT pathway. Pink and purple dots indicate proteins controlling macroautophagy; ATG8 proteins are represented in purple. ( B ) Proteins described in ( A ) are shown in a histogram of fold change. The hatched proteins are the common ones with the previously described HIV-associated proteome. ( C ) HeLa cells were transfected or not (NT non-transfected) with WT NL4-3 HIV-1 proviral DNA. Supernatants were collected, filtered and ultracentrifuged on a 20% sucrose cushion 48 h later. Western blot analysis of LC3B, GABARAP, GABARAPL1, GABARAPL2, TSG101, Beclin1, ATG5, Gag and CAp24 products, MAp17, Vpr, Vpu and Tubulin, in producing cells and supernatants. ( D ) LT CD4+ primary cells were infected, or not (NI, non-infected), with VSVg pseudotyped NL4-3 HIV-1 for 48 h at a MOI of 0.5. Western blot analysis of LC3B, GABARAP, GABARAPL1, GABARAPL2, TSG101, Beclin1, ATG5, Gag, and CAp24 products, MAp17, Vpr, Vpu, and Tubulin, in producing cells and supernatants. ( E ) HeLa cells were infected with VSVg pseudotyped NL4-3 HIV-1 at an MOI of 1. Supernatants were collected 48 h later, filtered and ultracentrifuged. IP of HIV-1 viral particles using human monoclonal anti-SUgp120 HIV-1 or human IgG1k as control. Western blot analysis of HIV-1 SUgp120, HIV-1 MAp17, LC3B, and GABARAPL1. ( F ) HeLa cells were infected with VSVg pseudotyped NL4-3 HIV-1 at an MOI of 1. Cell lysates were recovered 48 h later, and supernatants were collected, filtered and ultracentrifuged on a 20% sucrose cushion. Control (untreated) and subtilisin-treated supernatants were lysed, and the following proteins were analyzed by western blot: HIV-1 SUgp120, HIV-1 Gag and CAp24 products, LC3B, GABARAPL1 and GAPDH. All western blots are representative of at least three biological replicates. In all Western blots, LC3B-I, GABARAP-I, and GABARAPL1-I correspond to the non-lipidated form of these proteins and LC3B-II, GABARAP-II, and GABARAPL1-II to the lipidated forms. Data information: Statistical analysis in ( A , B ) was performed using a one-tailed paired Student t -test; ns not significant; n = 4 biological replicates. .

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal anti-CAp24 HIV-1 (NIBSC; EVA366), rabbit polyclonal anti-MAp17 HIV-1 (NIH; ARP-4811), rabbit polyclonal anti-Vpu HIV-1 (NIH; ARP-969), rabbit polyclonal anti-Vpr HIV-1 (NIH; ARP-3951), human monoclonal anti-SUgp120 HIV-1 (NIH; ARP-1476), rat monoclonal anti-HA-Peroxidase (Roche; 12013819001), mouse monoclonal anti-GAPDH (Santa Cruz; sc-47724), mouse monoclonal anti-tubulin (Sigma-Aldrich; T9026) mouse monoclonal anti-CD71 (TF-R) (Santa Cruz; sc-65882), rabbit polyclonal anti-ATG5 (Cell Signaling Technology; 12994S), rabbit polyclonal anti-Beclin1 (Santa Cruz; sc-11427), rabbit polyclonal anti-p62 (Life Technologies; PA5-27247), rabbit monoclonal anti-LC3A (Cell Signaling Technology; 4599 T; This antibody may also react with LC3B), rabbit polyclonal LC3B (Novus Biologicals; NB600-1384), rabbit monoclonal anti-GABARAP (Cell Signaling Technology; 13733 T), rabbit monoclonal anti-GABARAPL1 (Cell Signaling Technology; 26632 T), rabbit monoclonal anti-GABARAPL2 (Cell Signaling Technology; 14256 T), and mouse monoclonal anti-TSG101 (BD Biosciences; 612697).

Techniques: Transfection, Control, Liquid Chromatography with Mass Spectroscopy, Western Blot, Infection, One-tailed Test

Journal: EMBO Reports

Article Title: GABARAP proteins regulate the packaging of HIV-1 genomic RNA into virions

doi: 10.1038/s44319-025-00607-1

Figure Lengend Snippet: HeLa cells were transfected or not (NT) with WT, Udel, ΔVif, ΔVpr, and ΔNef NL4-3 HIV-1 proviral DNA for 48 h. Supernatants were collected and ultracentrifuged on a 20% sucrose cushion. Western blot analysis of LC3B, GABARAP, GABARAPL1, GABARAL2, HIV-1 SUgp120, HIV-1 Gag, and CAp24 products and GAPDH in producing cells and virion preparations. LC3B-I, GABARAP-I, and GABARAPL1-I correspond to the non-lipidated form of LC3B, GABARAP, and GABARAPL1, and LC3B-II, GABARAP-II, and GABARAPL1-II to the lipidated forms.

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal anti-CAp24 HIV-1 (NIBSC; EVA366), rabbit polyclonal anti-MAp17 HIV-1 (NIH; ARP-4811), rabbit polyclonal anti-Vpu HIV-1 (NIH; ARP-969), rabbit polyclonal anti-Vpr HIV-1 (NIH; ARP-3951), human monoclonal anti-SUgp120 HIV-1 (NIH; ARP-1476), rat monoclonal anti-HA-Peroxidase (Roche; 12013819001), mouse monoclonal anti-GAPDH (Santa Cruz; sc-47724), mouse monoclonal anti-tubulin (Sigma-Aldrich; T9026) mouse monoclonal anti-CD71 (TF-R) (Santa Cruz; sc-65882), rabbit polyclonal anti-ATG5 (Cell Signaling Technology; 12994S), rabbit polyclonal anti-Beclin1 (Santa Cruz; sc-11427), rabbit polyclonal anti-p62 (Life Technologies; PA5-27247), rabbit monoclonal anti-LC3A (Cell Signaling Technology; 4599 T; This antibody may also react with LC3B), rabbit polyclonal LC3B (Novus Biologicals; NB600-1384), rabbit monoclonal anti-GABARAP (Cell Signaling Technology; 13733 T), rabbit monoclonal anti-GABARAPL1 (Cell Signaling Technology; 26632 T), rabbit monoclonal anti-GABARAPL2 (Cell Signaling Technology; 14256 T), and mouse monoclonal anti-TSG101 (BD Biosciences; 612697).

Techniques: Transfection, Western Blot

Journal: EMBO Reports

Article Title: GABARAP proteins regulate the packaging of HIV-1 genomic RNA into virions

doi: 10.1038/s44319-025-00607-1

Figure Lengend Snippet: The mRNA quantity of LC3A was determined by RT-qPCR of total RNA extracted from HeLa cells or CD4 + T cells. HeLa cells or CD4 + T cells were infected or not (NI) with a VSVg pseudotyped WT NL4-3 HIV-1 at a MOI of 0.5 for 48 h. The mRNA quantity of LC3A was determined by RT-qPCR of total RNA extracted from cells. n = 2 biological replicates ( A ). HeLa cells transfected with control siRNA (siCTRL) or siRNA targeting LC3A, LC3B, LC3C, GABARAP, GABARAPL1, or GABARAPL2 were infected or not (NI) with a VSVg pseudotyped WT NL4-3 HIV-1 at a MOI of 0.5 for 48 h. The mRNA quantity of LC3B ( B ), LC3C ( C ), GABARAP ( D ), GABARAPL1 ( E ), or GABARAPL2 ( F ) was determined by RT-qPCR of total RNA extracted from cells. ( G ) Sequence alignment of LC3/GABARAP proteins using ESPript61. Identical residues are in red and similar ones are boxed. ( H ) Percentage of homology between LC3/GABARAP proteins. Data information: in ( B – F ), statistical analysis using a one-way ANOVA with Sidak’s multiple comparisons test; mean ± SEM; n = 3 biological replicates; ns not significant.

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal anti-CAp24 HIV-1 (NIBSC; EVA366), rabbit polyclonal anti-MAp17 HIV-1 (NIH; ARP-4811), rabbit polyclonal anti-Vpu HIV-1 (NIH; ARP-969), rabbit polyclonal anti-Vpr HIV-1 (NIH; ARP-3951), human monoclonal anti-SUgp120 HIV-1 (NIH; ARP-1476), rat monoclonal anti-HA-Peroxidase (Roche; 12013819001), mouse monoclonal anti-GAPDH (Santa Cruz; sc-47724), mouse monoclonal anti-tubulin (Sigma-Aldrich; T9026) mouse monoclonal anti-CD71 (TF-R) (Santa Cruz; sc-65882), rabbit polyclonal anti-ATG5 (Cell Signaling Technology; 12994S), rabbit polyclonal anti-Beclin1 (Santa Cruz; sc-11427), rabbit polyclonal anti-p62 (Life Technologies; PA5-27247), rabbit monoclonal anti-LC3A (Cell Signaling Technology; 4599 T; This antibody may also react with LC3B), rabbit polyclonal LC3B (Novus Biologicals; NB600-1384), rabbit monoclonal anti-GABARAP (Cell Signaling Technology; 13733 T), rabbit monoclonal anti-GABARAPL1 (Cell Signaling Technology; 26632 T), rabbit monoclonal anti-GABARAPL2 (Cell Signaling Technology; 14256 T), and mouse monoclonal anti-TSG101 (BD Biosciences; 612697).

Techniques: Quantitative RT-PCR, Infection, Transfection, Control, Sequencing

Journal: EMBO Reports

Article Title: GABARAP proteins regulate the packaging of HIV-1 genomic RNA into virions

doi: 10.1038/s44319-025-00607-1

Figure Lengend Snippet: ( A ) HeLa cells transfected with control siRNA (CTRL) or siRNA targeting LC3A, LC3B or LC3C were infected or not (NI) with a VSVg pseudotyped WT NL4-3 HIV-1 at a M.O.I. of 0.5. Cell lysates were recovered, and supernatants were collected 48 h later, filtered and ultracentrifuged on a 20% sucrose cushion. Western blot analysis of HIV-1 Gag and CAp24 products, LC3B and GAPDH in producing cells and supernatants. All western blots are representative of at least three biological replicates. In all western blot, LC3B-I, GABARAP-I, and GABARAPL1-I correspond to the non-lipidated form of these proteins and LC3B-II, GABARAP-II and GABARAPL1-II to the lipidated forms. ( B ) HeLa cells transfected with control siRNA (CTRL) or siRNA targeting GABARAP, GABARAPL1 or GABARAPL2 were infected with a VSVg pseudotyped WT NL4-3 HIV-1 at an MOI of 0.5. Cell lysates were recovered, and supernatants were collected 48 h later, filtered and ultracentrifuged on a 20% sucrose cushion. Western blot analysis of HIV-1 Gag and CAp24 products, GABARAP, GABARAPL1, GABARAPL2, and GAPDH in producing cells and supernatants. All western blots are representative of at least three biological replicates. HIV-1 release of viral production described in A and in B was calculated as the ratio between released CAp24 and cell-associated CAp24 quantified by ELISA, in ( C , D ), respectively. Results were normalized to control cells. The infectivity of the released virus in productions described in ( A , B ) was determined by a β-galactosidase reporter assay and normalized by the quantity of released CAp24, in ( E , F ), respectively. Data information: In ( C – F ), statistical analysis was performed using a one-way ANOVA with Dunnett’s multiple comparisons test; mean ± SEM; n = 3 biological replicates; ns not significant. .

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal anti-CAp24 HIV-1 (NIBSC; EVA366), rabbit polyclonal anti-MAp17 HIV-1 (NIH; ARP-4811), rabbit polyclonal anti-Vpu HIV-1 (NIH; ARP-969), rabbit polyclonal anti-Vpr HIV-1 (NIH; ARP-3951), human monoclonal anti-SUgp120 HIV-1 (NIH; ARP-1476), rat monoclonal anti-HA-Peroxidase (Roche; 12013819001), mouse monoclonal anti-GAPDH (Santa Cruz; sc-47724), mouse monoclonal anti-tubulin (Sigma-Aldrich; T9026) mouse monoclonal anti-CD71 (TF-R) (Santa Cruz; sc-65882), rabbit polyclonal anti-ATG5 (Cell Signaling Technology; 12994S), rabbit polyclonal anti-Beclin1 (Santa Cruz; sc-11427), rabbit polyclonal anti-p62 (Life Technologies; PA5-27247), rabbit monoclonal anti-LC3A (Cell Signaling Technology; 4599 T; This antibody may also react with LC3B), rabbit polyclonal LC3B (Novus Biologicals; NB600-1384), rabbit monoclonal anti-GABARAP (Cell Signaling Technology; 13733 T), rabbit monoclonal anti-GABARAPL1 (Cell Signaling Technology; 26632 T), rabbit monoclonal anti-GABARAPL2 (Cell Signaling Technology; 14256 T), and mouse monoclonal anti-TSG101 (BD Biosciences; 612697).

Techniques: Transfection, Control, Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Virus, Reporter Assay

Journal: EMBO Reports

Article Title: GABARAP proteins regulate the packaging of HIV-1 genomic RNA into virions

doi: 10.1038/s44319-025-00607-1

Figure Lengend Snippet: ( A ) Parental, LC3 (LC3 TKO), or GABARAP (GABARAP TKO) knock down cells were incubated in full medium (FM), full medium with Bafilomycin A1 ( B ), EBSS for amino acid depletion without or with Bafilomycin A1 (ES and EB, respectively) for 2 h. Western blot analysis of LC3A/B, LC3B, GABARAP, GABARAPL1, GABARAPL2, and GAPDH in cells. LC3A/B-I, GABARAP-I, and GABARAPL1-I correspond to the non-lipidated form of LC3A/B, GABARAP and GABARAPL1, and LC3A/B-II, GABARAP-II and GABARAPL1-II to the lipidated forms. All western blots are representative of at least three biological replicates. ( B ) Parental or ATG5−/− 92#3 knockout HeLa cells were transfected or not (NT) with WT NL4-3 HIV-1 proviral DNA for 48 h. Cell lysates were recovered, and supernatants were collected, filtered, and ultracentrifuged on a 20% sucrose cushion. Western blot analysis of LC3B, GABARAP, GABARAPL1, GABARAPL2, HIV-1 Gag and CAp24 products, ATG5 and GAPDH in producing cells and virion preparations. All western blots are representative of at least three biological replicates. ( C ) The infectivity of released virus was determined by a β-galactosidase reporter assay and normalized by the quantity of released CAp24. Data information: Statistical analysis using a one-tail unpaired Student’s t- test; mean ± SEM; n = 3 biological replicates; ns not significant.

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal anti-CAp24 HIV-1 (NIBSC; EVA366), rabbit polyclonal anti-MAp17 HIV-1 (NIH; ARP-4811), rabbit polyclonal anti-Vpu HIV-1 (NIH; ARP-969), rabbit polyclonal anti-Vpr HIV-1 (NIH; ARP-3951), human monoclonal anti-SUgp120 HIV-1 (NIH; ARP-1476), rat monoclonal anti-HA-Peroxidase (Roche; 12013819001), mouse monoclonal anti-GAPDH (Santa Cruz; sc-47724), mouse monoclonal anti-tubulin (Sigma-Aldrich; T9026) mouse monoclonal anti-CD71 (TF-R) (Santa Cruz; sc-65882), rabbit polyclonal anti-ATG5 (Cell Signaling Technology; 12994S), rabbit polyclonal anti-Beclin1 (Santa Cruz; sc-11427), rabbit polyclonal anti-p62 (Life Technologies; PA5-27247), rabbit monoclonal anti-LC3A (Cell Signaling Technology; 4599 T; This antibody may also react with LC3B), rabbit polyclonal LC3B (Novus Biologicals; NB600-1384), rabbit monoclonal anti-GABARAP (Cell Signaling Technology; 13733 T), rabbit monoclonal anti-GABARAPL1 (Cell Signaling Technology; 26632 T), rabbit monoclonal anti-GABARAPL2 (Cell Signaling Technology; 14256 T), and mouse monoclonal anti-TSG101 (BD Biosciences; 612697).

Techniques: Knockdown, Incubation, Western Blot, Knock-Out, Transfection, Infection, Virus, Reporter Assay

Journal: EMBO Reports

Article Title: GABARAP proteins regulate the packaging of HIV-1 genomic RNA into virions

doi: 10.1038/s44319-025-00607-1

Figure Lengend Snippet: Parental, LC3 (LC3 TKO), or GABARAP (GABARAP TKO) knockout cells were transfected or not (NT) with WT NL4-3 HIV-1 proviral DNA. Cell lysates were recovered, and supernatants were collected 48 h later, filtered and ultracentrifuged on a 20% sucrose cushion. ( A ) Western blot analysis of LC3B, GABARAPL1, HIV-1 SUgp120, HIV-1 Gag, and CAp24 products and GAPDH in producing cells and supernatants. All western blots are representative of at least three biological replicates. LC3B-I and GABARAPL1-I correspond to the non-lipidated form of LC3B and GABARAPL1, and LC3B-II and GABARAPL1-II to the lipidated forms. Intracellular ( B ) and extracellular ( C ) CAp24 were quantified by ELISA. Results were normalized to the control condition. ( D ) HIV-1 release was calculated as the ratio between released CAp24 and cell-associated CAp24 quantified by ELISA. Results were normalized to the control condition. ( E ) The infectivity of the released virus was determined by a β-galactosidase reporter assay and normalized by the quantity of released CAp24. Results were normalized to the control condition. Data information: statistical analysis was performed using a one-way ANOVA with Dunnett’s multiple comparisons test; mean ± SEM; n = 7 biological replicates. ns not significant. .

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal anti-CAp24 HIV-1 (NIBSC; EVA366), rabbit polyclonal anti-MAp17 HIV-1 (NIH; ARP-4811), rabbit polyclonal anti-Vpu HIV-1 (NIH; ARP-969), rabbit polyclonal anti-Vpr HIV-1 (NIH; ARP-3951), human monoclonal anti-SUgp120 HIV-1 (NIH; ARP-1476), rat monoclonal anti-HA-Peroxidase (Roche; 12013819001), mouse monoclonal anti-GAPDH (Santa Cruz; sc-47724), mouse monoclonal anti-tubulin (Sigma-Aldrich; T9026) mouse monoclonal anti-CD71 (TF-R) (Santa Cruz; sc-65882), rabbit polyclonal anti-ATG5 (Cell Signaling Technology; 12994S), rabbit polyclonal anti-Beclin1 (Santa Cruz; sc-11427), rabbit polyclonal anti-p62 (Life Technologies; PA5-27247), rabbit monoclonal anti-LC3A (Cell Signaling Technology; 4599 T; This antibody may also react with LC3B), rabbit polyclonal LC3B (Novus Biologicals; NB600-1384), rabbit monoclonal anti-GABARAP (Cell Signaling Technology; 13733 T), rabbit monoclonal anti-GABARAPL1 (Cell Signaling Technology; 26632 T), rabbit monoclonal anti-GABARAPL2 (Cell Signaling Technology; 14256 T), and mouse monoclonal anti-TSG101 (BD Biosciences; 612697).

Techniques: Knock-Out, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Infection, Virus, Reporter Assay

Journal: EMBO Reports

Article Title: GABARAP proteins regulate the packaging of HIV-1 genomic RNA into virions

doi: 10.1038/s44319-025-00607-1

Figure Lengend Snippet: ( A ) HA-trap Immunoprecipitation (IP) in HeLa cells extract co-transfected with plasmids expressing WT NL4-3 HIV-1 proviral DNA and pCMV-HA, HA-GABARAP, -GABARAPL1, or -GABARAPL2. Immunoprecipitated proteins were detected by western blotting of HIV-1 Gag and CAp24 products, HA and GAPDH. The higher band of HA-tagged GABARAP proteins corresponds to their non-lipidated form, and the lower corresponds to their lipidated form. All western blots are representative of at least three biological replicates. ( B ) HA-trap IP in HeLa cells, treated or not with RNase, extracts co-transfected with plasmids expressing WT NL4-3 HIV-1 proviral DNA and HA-GABARAPL1 or HA-LC3B. Immunoprecipitated proteins were detected by western blotting of HIV-1 Gag and CAp24 products, HA and GAPDH. All western blots are representative of at least three biological replicates. ( C ) Quantification of the ratio between Pr55Gag and HA protein levels in ( B ). Statistical analysis using an unpaired Student’s t -test; mean ± SEM; n = 4 biological replicates. ( D ) HeLa parental cells were infected with VSVg-pseudotyped HIV-1 viruses at a MOI of 1. Forty-eight hours later, cells were UV-crosslinked. RNA was immunoprecipitated with antibodies against GABARAPL1 or IgG control. Viral gRNA from RIP were quantified by RT-qPCR and normalized to inputs and control immunoprecipitation. Statistical analysis using a one-way ANOVA with Sidak’s multiple comparisons test; mean ± SEM; n = 6 biological replicates. ( E ) Western blot analysis from RIP in ( D ) of GABARAPL1. GABARAPL1-I corresponds to the non-lipidated form of GABARAPL1 and GABARAPL1-II to the lipidated form. All western blots are representative of at least three biological replicates. ( F ) Parental HeLa and GABARAP TKO cells were infected with VSVg-pseudotyped HIV-1 viruses at an MOI of 1. Forty-eight hours later, cells were UV-crosslinked. RNA was immunoprecipitated with antibodies against Gag or serum control. Viral gRNA from RIP were quantified by RT-qPCR and normalized to inputs and control immunoprecipitation. Statistical analysis using a one-way ANOVA with Sidak’s multiple comparisons test; mean ± SEM; n = three biological replicates. ( G ) Western blot analysis from RIP in ( F ) of HIV-1 Gag and CAp24 products. All western blots are representative of at least three biological replicates. .

Article Snippet: The following antibodies were used for immunoblotting: mouse monoclonal anti-CAp24 HIV-1 (NIBSC; EVA366), rabbit polyclonal anti-MAp17 HIV-1 (NIH; ARP-4811), rabbit polyclonal anti-Vpu HIV-1 (NIH; ARP-969), rabbit polyclonal anti-Vpr HIV-1 (NIH; ARP-3951), human monoclonal anti-SUgp120 HIV-1 (NIH; ARP-1476), rat monoclonal anti-HA-Peroxidase (Roche; 12013819001), mouse monoclonal anti-GAPDH (Santa Cruz; sc-47724), mouse monoclonal anti-tubulin (Sigma-Aldrich; T9026) mouse monoclonal anti-CD71 (TF-R) (Santa Cruz; sc-65882), rabbit polyclonal anti-ATG5 (Cell Signaling Technology; 12994S), rabbit polyclonal anti-Beclin1 (Santa Cruz; sc-11427), rabbit polyclonal anti-p62 (Life Technologies; PA5-27247), rabbit monoclonal anti-LC3A (Cell Signaling Technology; 4599 T; This antibody may also react with LC3B), rabbit polyclonal LC3B (Novus Biologicals; NB600-1384), rabbit monoclonal anti-GABARAP (Cell Signaling Technology; 13733 T), rabbit monoclonal anti-GABARAPL1 (Cell Signaling Technology; 26632 T), rabbit monoclonal anti-GABARAPL2 (Cell Signaling Technology; 14256 T), and mouse monoclonal anti-TSG101 (BD Biosciences; 612697).

Techniques: Immunoprecipitation, Transfection, Expressing, Western Blot, Infection, Control, Quantitative RT-PCR

Journal: Genes & Development

Article Title: Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

doi: 10.1101/gad.276428.115

Figure Lengend Snippet: Disruption of FIP200 interaction with Atg13 blocks autophagy in MEFs. ( A ) Schematic representation of the FIP200-4A mutant knock-in targeting vector and the targeted allele of the FIP200-4A mutant. Solid boxes represent regions of vector homology with the target locus; thick, numbered boxes represent the exons flanking homology regions. Site-directed mutagenesis was used to introduce the LQFL-to-AAAA mutation at the 582–585 amino acid residues. The asterisk denotes the mutated residues on exon 13. (EcoRV) EcoRV sites; (neo) neomycin-resistant gene cassette; (DTA) diphtheria toxin gene. P1 and P2 are a pair of primers for genotyping. P3 and P4 are a pair of primers for sequencing amplified genomic DNA with mutations. ( B ) Genomic DNA was extracted from mouse tails and analyzed by PCR using P1 and P2 to detect the FIP200 wild-type allele (178 base pairs [bp]) or knock-in allele (448 bp). Representative genotypes of a litter of mice from crosses of FIP200 +/ KI mice with wild-type B6 mice. ( C ) Representative genotypes of MEF cells prepared from embryonic day 13.5 (E13.5) embryos of intercrosses of FIP200 +/ KI mice. ( D ) Lysates from different MEFs were analyzed by immunoblotting using antibodies against various proteins as indicated. ( E ) MEFs were incubated in fresh growth medium (control) or HBSS (starvation) in the presence of 100 nM BafA1 for 2 h. Lysates were then prepared and analyzed by immunoblotting using antibodies against proteins as indicated. ( F ) FIP200 +/+ and FIP200 KI/KI MEFs were starved in HBSS for 2 h, fixed, and stained for endogenous LC3B. Representative images are shown. Bar, 10 μm. ( G ) Quantification of LC3B puncta number in starved MEF cells. At least 50 cells per experiment were counted. (**) P < 0.01. ( H ) Representative transmission electron microscopy images of starved MEF FIP200 +/+ and MEF FIP200 KI/KI cells. Arrows indicate autophagosomal structures ( left panel) and mitochondria ( right panel), respectively. Bars, 500 nm. ( I ) Genotypes of progeny from FIP200 +/ KI intercrosses.

Article Snippet: Primary antibodies against LC3B (1:200; Cell Signaling, 3868) were used.

Techniques: Disruption, Mutagenesis, Knock-In, Plasmid Preparation, Introduce, Sequencing, Amplification, Western Blot, Incubation, Control, Staining, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

doi: 10.1038/s41598-017-06319-4

Figure Lengend Snippet: Co-immunoprecipitation (IP) of the Nef-ATG8 complex. Co-IP studies were carried out with lysates prepared from HEK293 cells stably expressing Nef-DsRed ( A–C ) and cotransfected with plasmids encoding for the indicated YFP-ATG8 constructs ( D–F ). ( A ) and ( D ) show samples before immunoprecipitation. In ( A ) an anti-GABARAPL2 (a), an anti-GABARAP (b), an anti-GABARAPL1 (c), and an anti-LC3B antibody (d) were used to detect the different ATG8s. ( B ) Immunoprecipitates with anti-DsRed antibody followed by SDS-PAGE and immunoblotting with various ATG8 antibodies (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B). ( C ) In a reciprocal set of experiments, immunoprecipitates were carried out with various ATG8 (a: anti-GABARAPL2, b: anti-GABARAP, c: anti-GABARAPL1 and d: anti-LC3B) antibodies followed by SDS-PAGE and immunoblotting with an anti-DsRed antibody. In ( D ) an anti-DsRed antibody was used to detect Nef fused to DsRed and an anti-YFP antibody to detect YFP and the ATG8s fused to YFP. ( E ) Immunoprecipitates with an anti-DsRed antibody or ( F ) with an anti-GFP antibody followed by SDS-PAGE and immunoblotting with the antibodies are indicated. Data are representative of three independent experiments (U: unbound material; E: eluate fraction). Full-length western blots are presented in Supplementary Figs and .

Article Snippet: Rabbit polyclonal antibodies raised against GABARAP (18723-1-AP), GABARAPL2 (18724-1-AP), GABARAPL1 (18721- 1-AP), and LC3B (18725-1-AP) were obtained from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Construct, SDS Page, Western Blot

Journal: Scientific Reports

Article Title: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

doi: 10.1038/s41598-017-06319-4

Figure Lengend Snippet: GABARAP residues S53 and S62 are essential for Nef binding. ( A ) Human ATG8s sequence alignment indicates putative key residues for Nef binding specificity. Protein names of ATG8s showing Nef binding during pull-down and immunoprecipitation analysis are given in different shades of yellow, and include all members of the GABARAP subfamily. Protein names of LC3B and of the other LC3s are given in different shades of brown. Purple arrows indicate residues of GABARAP showing chemical shift changes higher than 0.05 ppm (see Fig. ) upon Nef titration. Residues forming HP1 and HP2 are shaded in light and dark blue, respectively. Red asterisks highlight putative key positions for determining Nef-binding specificity. The corresponding amino acids found in GABARAP and LC3B at these positions are highlighted in red. ( B ) Visualization of HP1 and HP2 on the surface of the GABARAP structure [PDB ID: 1KOT]. ( C ) Structural overlay of GABARAP and LC3B with the putative key residues necessary for Nef-binding highlighted. Cartoon representations of GABARAP (yellow) and LC3B (brown) [PDB ID: 1V49] showing their regular secondary structure elements demonstrate that S53 and F62 of GABARAP as well as the corresponding D56 and K65 of LC3B are surface exposed, and therefore are available for ligand binding. Details highlight the side chain orientations of the key residues, for this the structures have been rotated appropriately. ( D ) Nef-conjugated or free Sepharose beads (control) were incubated with GABARAP(S53D/F62K) or LC3B(D56S/K65F) mutants. The input, the unbound material of the flow through (U),the wash (W) fractions and the eluate (E) fractions were subjected to SDS-PAGE and visualized by CBB staining. Full-length gels are presented in Supplementary Fig. .

Article Snippet: Rabbit polyclonal antibodies raised against GABARAP (18723-1-AP), GABARAPL2 (18724-1-AP), GABARAPL1 (18721- 1-AP), and LC3B (18725-1-AP) were obtained from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Titration, Ligand Binding Assay, Control, Incubation, SDS Page, Staining

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 G425R ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length GST-SQSTM1 sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: In Vitro, Sequencing, Isolation, Incubation, Western Blot

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: ESI-MS indicates weaker binding of the LIR (L341V) to LC3B compared to WT LIR. (A) Native ESI-MS spectrum of an equimolar mixture of WT LIR and LIR (L341V) peptides (5 µM, residues 332 to 351). (B) LIR peptide mixture titrated with 5 µM LC3B. Top, full spectrum indicating free LIR (gray filled circle, mixture of WT LIR and LIR [L341V]), free LC3B and LC3B-LIR complexes of indicated (multiple) charge states. Below, zoomed-in spectra showing m/z values of the free and bound LIR complexes at the indicated charge states.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Binding Assay

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: Isothermal titration calorimetry (ITC) binding isotherms from titrating LC3B with LIR peptides at 25°C. Binding isotherms were fitted to a one-site binding model and K d values determined. Starting concentration of the LC3B was approximately 30 µM, and of LIR peptide (residues 332 to 351) stocks approximately 350 µM. Black diamond, LIR (L341V); gray circle WT LIR.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Isothermal Titration Calorimetry, Binding Assay, Concentration Assay

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: NMR titrations indicate selective perturbations of LC3B residues upon binding of LIR peptides. (A) 1 H- 15 N-HSQC spectrum of LC3B (0.25 mM dark gray) overlaid with the spectrum of the complex of LC3B with WT LIR (0.5 mM blue, residues 332 to 351) (ratio of 1:2) showing extensive chemical shift perturbations (CSPs) across the spectrum induced by ligand binding at 298 K; (B) Expansion of the region highlighted in (A) showing overlap between the spectrum of LC3B (dark gray), LC3B+WT LIR (blue) and LC3B + LIR (L341V) (green) illustrating a number of key residues within the LIR binding pocket that are perturbed to different extents by the WT LIR and LIR (L341V). Arrows identify the shifts of V33, F52 and V54 in the 2 complexes, while the majority of other residues show only small differences between the spectra of the 2 complexes (overlap of blue and green peaks), demonstrating selective effects on residues in direct contact with the LIRs.

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Binding Assay, Ligand Binding Assay

Journal: Autophagy

Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

doi: 10.1080/15548627.2016.1170257

Figure Lengend Snippet: Chemical shift mapping of the binding of the WT LIR and LIR (L341V) peptides (residues 332 to 351) to 15 N-LC3B. (A) Chemical shift mapping of the WT LIR. Weighted chemical shift pertubation (CSP) data showing the residues of 15 N-LC3B (0.25 mM) that are perturbed upon WT LIR binding at a final ratio of 1:2 (LC3B:LIR) at 298 K. All CSPs above 1.0 are indicated. (B) Difference in CSP effects between WT LIR and LIR (L341V) sequences from NMR titrations at a final ratio of 1:2 (LC3B:LIR) at 298 K. The indicated residues showed CSPs that are substantially different between the 2 complexes (greater in the WT LIR). (C) Representation of binding surface of LC3B with WT LIR, generated by highlighting residues determined from NMR chemical shift mapping experiments. LC3B residues in blue showed the greatest CSP values. Backbone representation of the LIR peptide also indicated (residues DDDWTHLS, 335 to 342 shown). (D) Residues showing substantially different CSPs from the NMR titrations of LC3B with WT LIR compared to LIR (L341V), highlighted on the LC3B structure. Note the close correlation with the binding cleft in proximity to L341 of the LIR peptide (site of L341V indicated).

Article Snippet: One ml of each diluted lysate was incubated at 37°C with excess glutathione-Sepharose (GE Healthcare, 17-0756-01), ubiquitin-Sepharose (10 mg/ml human ubiquitin immobilized on CNBr-activated Sepharose 4B [Sigma-Aldrich, C9142]), LC3B-Sepharose (1 mg/ml LC3B immobilized on CNBr-activated Sepharose 4B) or control-Sepharose (CNBr-activated Sepharose prepared without ubiquitin or LC3B).

Techniques: Binding Assay, Generated

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Myc, GFP, or 3xFlag tag is fused to human MAP1LC3B ORF at its N- terminus. The box bars on the left represent the N-terminal tags. The dashed lines represent MAP1LC3B with 27 amino acids sequence shown at the carboxyl terminus. The arrow points to Glycine 120 conjugation site. The last five amino acids at the C-terminal are underlined. Mutant amino acid or last amino acid of LC3B is bolded. Human LC3C, Rat LC3, Rat LC3B, Mouse LC3A, Mouse LC3B and the related mutants are described in Result section.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Sequencing, Conjugation Assay, Mutagenesis

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Aa , plasmids expressing Myc-LC3B, myc-LC3B G120A or Myc-LC3B G120 was expressed in HEK293 cells for 24 hours. Cells were harvested and lysed by sonication, and 10 µg of total protein of each lysate were separated by 15% of SDS-polyacrylamide gel, followed by immunoblotting with anti-LC3B antibody. Ab, plasmids expressing Flag-LC3B or Flag-LC3B G120A was expressed in HEK293 cells for 24 hours. Cells were lysed followed by immnuoblotting using Flag-antibody. B, plasmids expressing LC3B, LC3B G120A or LC3B A120 was expressed in HEK293 cells for 24 hours. Cells were treated with 50 µM of CQ for 2 hours before collection. Transient expressed proteins were analyzed by immunoblotting using LC3B antibody. Ca, plasmids expressing GFP-LC3B or GFP-LC3 G120A was expressed in HEK293 cells for 24hours. Cells were then treated with 50 µM of Chloroquine (CQ) for 2 hours. Wb assays were performed using anti-GFP antibody. Cb, A549 cells infected with Adenoviral vectors expressing GFP-LC3B or GFP-LC3B G120A at 5 MOI for 24 hours. Cells were then treated with 50 µM of CQ for two hours. GFP-LC3B punctuation was recorded by fluorescence microscopy. CM, complete media, CQ, complete media plus CQ. Scale bar: 25 micron.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Expressing, Sonication, Western Blot, Infection, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: A, plasmids expressing Flag-LC3B, Flag-LC3B G120A , Flag-LC3B G120 or Flag-LC3 A120 was expressed in HEK293 cells for 24 hours. Cells were treated with 50 µM of CQ for two hours and harvested and lysed. Total cell lysates (10 µg) was used for immunoblotting by Flag antibody. B, G120 point mutant plasmids were expressed in HEK293 cells for 24 hours. Immunoblotting was conducted to compare the band’s mobility with Flag-LC3B, Flag-LC3B G120A or Flag-LC3B G120A as indicated. C, Flag-LC3B G120D and Flag-LC3B G120E were expressed in HEK293 cells for 24 hours. Cells were then treated with 50 µM of CQ for 2 hours. Expression patters of Flag-LC3B G120D and Flag-LC3B G120E were compared with endogenous LC3B by immunoblotting with LC3B antibody (upper panels). Adenoviral vector expressing GFP-LC3B, GFP-LC3B G120D or GFP-LC3B G120E was used to infect A549 cells for 24 hours. Cells were then treated with 50 µM of CQ for 2 hours. Images were recorded using fluorescence microscopy. Scale bar: 25 micron. D, Truncates of LC3B (see Fig. 1) were expressed in HEK293 for 24hours. Cells were treated with 50 µM of CQ for two hours before harvesting. Total cell lysates were used for Wb assay to compare the mobility with Flag-LC3B-I and Flag-LC3B-II. Flag antibody was used to detect the recombinant proteins.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Expressing, Western Blot, Mutagenesis, Plasmid Preparation, Fluorescence, Microscopy, Recombinant

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Flag-LC3B G120A expression vector was transfected into HEK293 cell for 48 hours. Cells were harvested and lysed with protease inhibitor cocktail. The lysates were centrifuged at 10,000 g for 30 min. Supernatants were then used for affinity purification of Flag-LC3B G120A by Flag antibody conjugated beads in column according to the proposed protocol. A, 20 µl of elution (from total 1 ml) from Flag antibody based affinity column was loaded onto 12.5% of SDS-polyacrylamide gel. Gel was stained with PageBlue Protein Staining Solution. Elution number 2 and 3 were combined and concentrated. Purified Flag-LC3B G120A was used to run MALDI ISD. Intact mass spectra are shown in B and in-source decay spectra are shown in C . For database searching selected ISD fragments (* in the spectra) were used as virtual precursors and submitted to the Mascot search engine MS/MS ion search program (Matrix Science, Ltd. Version 2.3) through the Shimadzu Biotech Launchpad software. D, peptide view of Mascot Search results MS/MS Fragmentation of N-terminal: MDYKDHDGDYKDHDIDYKDDDDKLQVDPSEKTFKQRRTFEQRVED (Found in IPI00953817; Data file: Automatically uploaded data; Average mass of neutral peptide Mr(calc): 5622.8801; Ions Score: 168 Expect: 2.1e-012; Matches : 28/88 fragment ions using 47 most intense peaks.). E, peptide view of Mascot Search results: MS/MS Fragmentation of C-terminal: LLVNGHSMVSVSTPISEVYESEKDEDGFLYMVYASQETFAMKLSV(Found in IPI00953817; Data file Automatically uploaded data; Average mass of neutral peptide Mr(calc): 5017.6143; Ions Score: 34 Expect: 53;Matches : 30/88 fragment ions using 134 most intense peaks.). F, Theoretical fragment ions for the peptide sequence. The matched fragment ions are highlighted in red typed. G, Protein view: Match to: IPI00953817 Score: 297. Found in search of Automatically uploaded data. Nominal mass (M r ): 17870; Calculated pI value: 5.38. NCBI BLAST search of IPI00953817 against nr Unformatted sequence string for pasting into other applications. Sequence Coverage: 59%. Matched peptides shown in Bold Red. The N-terminal 3xFlag tag and C-terminal last five amino acids of Flag-LC3B G120A are underlined. The downward pointing arrow shows the LC3B start amino acid in the recombinant protein (first amino acid methionine was removed in the fusion protein). The arrow pointing upward shows the G120A point mutation in the Flag-LC3B G120A .

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Expressing, Plasmid Preparation, Transfection, Protease Inhibitor, Affinity Purification, Affinity Column, Staining, Purification, Tandem Mass Spectroscopy, Software, Sequencing, Recombinant, Mutagenesis

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: A, pTriEX.1.1 plasmids encoding Flag-LC3B, Flag-LC3B G120A , or Flag-LC3B A120 was expressed in HEK293 or E.coli cells (Tuner cell), respectively. Recombinant protein expression in E.coli cells were induced with 1 mM of IPTG for four hours before harvesting. 10 µg of HEK293 or E.coli lysates were loaded onto SDS-polyacrylamide gel. Immunoblotting was conducted using Flag-antibody. B, 10 µg of E.coli lysates with Flag-LC3B or Flag-LC3B G120A recombinant proteins was digested with recombinant Atg4B at 37°C for indicated time and analyzed by immunoblotting with LC3B antibody and Atg4B antibody as indicated.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Recombinant, Expressing, Western Blot

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: A, Flag-LC3B and indicated mutants were expressed in E.coli. 10 µg of cell lysates were then separated in 15% of SDS-polyacrylamide gel. Transferred membrane was stained with Ponceau S before immunoblotting with LC3B antibody. B, human LC3B and indicated mutants were expressed in HEK293 cells for 24 hours. Cells were then treated with 50 µM of CQ for 2 hours. Expression patters were analyzed by Wb using LC3B antibody. C, Same sets of expression plasmids were then expressed in E.coli cells. 10 µg of cell lysates were then separated in 15% of SDS-polyacrylamide gel. Transferred membrane was stained with Ponceau S before immunoblotting with LC3B antibody. Arrows point to wild-type and mutant LC3B recombinant protein band expressed in E. coli.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Membrane, Staining, Western Blot, Expressing, Mutagenesis, Recombinant

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: Pairwise alignments of LC3A, LC3B and LC3C of human and rodent origin are generated by ClustalW2. The glycine conjugation sites are boxed and C-terminal amino acids after glycine conjugation site are underlined. A “*” presents identity of amino acids; “ : ” presents these amino acids in a same amino acid group (similarity); “⋅” presents different group of amino acids.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Generated, Conjugation Assay

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: A, Compare pro-human LC3B with pro-mouse LC3B, pro-rat LC3B and their cleaved and lipidated forms’ band patterns by 15% of SDS-polyacrylamide gel. Indicated expression plasmids were expressed in HEK293 cells. B, compare pro-rat LC3B with pro-rat LC3 and cleaved forms by 15% of SDS-polyacrylamide gel. Correspondent expression plasmids were expressed in HEK293 cells. Note: human LC3B-I band is close to RatLC3B-I/RatLC3-I as indicated by arrows. C, pro-LC3A and its cleaved form comparison by 15% of SDS-polyacrylamide gel. Indicated expression plasmids were expressed in HEK293 cells. D, compare pro-human LC3B and LC3C species by 15% of SDS-polyacrylamide gel. Indicated expression plasmids were expressed in HEK293 cells for 24 h. HEK293 cells transient expressing Human LC3C were treated with 50 µM of CQ for 2 h to induce accumulation of human LC3B-II and LC3C-II. Total cell lysates were separated by a 15% of SDS-polyacrylamide gel. Protein transferred membrane was cut between human LC3B group and human LC3C group and immunoblotting by LC3B and LC3C antibody, respectively. The immunoblotting membrane was re-aligned before developing with chemiluminescent substrate in order to compare human LC3B species and human LC3C species in Wb.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Expressing, Comparison, Membrane, Western Blot

Journal: PLoS ONE

Article Title: The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

doi: 10.1371/journal.pone.0074222

Figure Lengend Snippet: A, MEFwt and MEFatg7KO cells were treated with 50 µM of CQ for 2 hours. LC3B expression patterns were then analyzed in 15% of SDS-PAGE by immunoblotting with LC3B antibody. B, Adenoviral vector expressing GFP-LC3B was used to infect MEFatg7KO cells in series double diluted doses for 24 hours (D1 = 20MOI). Ad-GFP-LC3B (20MOI) infected wild-type MEF cells were used as control or treated with 50 µM of CQ for 2 hours.GFP-LC3B expression patterns were analyzed by 15% of SDS-PAGE. C, Ad-GFP-LC3B infected MEFwt and MEFatg7KO cells were treated with 50 µM of CQ for 2 hours and images were recorded by fluorescence microscopy. D, HEK293 cells were transfected with Atg4B siRNA and Control siRNA for 48 h. 15 µg of total cell lysates were analyzed by immunoblotting with Atg4B, LC3B, and β-actin antibodies. Image J software was used to quantify band density using β-actin as loading control. E, Diagram of LC3 species migration patterns in 15% of SDS-polyacrylamide gel. The diagram summarizes LC3 species (pro-LC3, LC3-I and LC3-II) band patterns from human, mouse and rat origins. Refer to the result section for description in details.

Article Snippet: The following antibodies were used: Rabbit anti- LC3B antibody (Cell Signaling, raised by a synthetic peptide from N-terminal 2-25aa); Rabbit anti- LC3A antibody (abcam, raised by a synthetic peptide from N-terminal 2-15aa); Rabbit anti- LC3C antibody (Abnova, raised by a synthetic peptide from N-terminal 2-30aa); mouse anti-GFP antibody (Santa Cruz biotech); mouse anti- FLAG M2 and β-actin antibodies (Sigma-Aldrich); and Goat anti-rabbit IgG (HRP-conjugated) and Goat anti-mouse IgG (HRP-conjugated) (Jackson ImmunoResearch).

Techniques: Expressing, SDS Page, Western Blot, Plasmid Preparation, Infection, Control, Fluorescence, Microscopy, Transfection, Software, Migration

Journal: Cell & Bioscience

Article Title: Chorionic villus-derived mesenchymal stem cell-mediated autophagy promotes the proliferation and invasiveness of trophoblasts under hypoxia by activating the JAK2/STAT3 signalling pathway

doi: 10.1186/s13578-021-00681-7

Figure Lengend Snippet: CV-MSCs promote the autophagy, proliferation and invasion of trophoblasts in vitro. A Representative CCK-8 assay results for JAR, JEG-3 and HTR-8 cells are shown. Trophoblast cells were treated with CV-MSC CM under hypoxic conditions. B Representative Transwell assay results for trophoblasts are shown. The number of trophoblasts that migrated through the 8-μm Transwell membrane pores was counted to determine changes in the invasive capabilities in response to CV-MSC CM under hypoxic conditions (scale bar, 50 μm). C Transmission electron microscopy (TEM) analysis showed autophagosomes in the cytoplasm of trophoblast cells treated with CV-MSC CM under hypoxic conditions (scale bar, 1 μm). D Whole cell lysates from trophoblast cells were subjected to western blotting to analyse LC3, BECN1, p62, MMP2 and MMP9 levels. β-actin was included as a loading control. ***P < 0.001

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human JAK2, pJAK2, STAT3, pSTAT3, MMP2, MMP9, LC3, BECN1 and P62 (1:1000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL) were incubated with the blocked membranes.

Techniques: In Vitro, CCK-8 Assay, Transwell Assay, Membrane, Transmission Assay, Electron Microscopy, Western Blot, Control

Journal: Cell & Bioscience

Article Title: Chorionic villus-derived mesenchymal stem cell-mediated autophagy promotes the proliferation and invasiveness of trophoblasts under hypoxia by activating the JAK2/STAT3 signalling pathway

doi: 10.1186/s13578-021-00681-7

Figure Lengend Snippet: Global transcriptome characterization of trophoblasts regulated by CV-MSCs; JAK2/STAT3 signalling pathway activation by CV-MSCs mediates increased trophoblast autophagy. A Heat map of some differentially expressed genes in JEG-3 cells treated with or without CV-MSC CM under hypoxic conditions according to RNA-seq. B GSEA of the most upregulated and downregulated pathways in JEG-3 cells treated with CV-MSC CM compared with JEG-3 cells cultured alone. C Significant increases in STAT3, IL-6 and LIF mRNA levels were found in JAR, JEG-3 and HTR-8 cells treated with CV-MSC CM by qRT-PCR. D Significant increases in p-JAK2 and p-STAT3 were found in JAR, JEG-3 and HTR8 cells treated with CV-MSC CM under hypoxic conditions by western blotting. E Immunoblot analysis of IL-6 and LIF protein levels in normal CV-MSCs, PE-CV-MSCs and CV-MSCs under hypoxia condition. F Three trophoblast cell lines were cultured with normal CV-MSC CM or PE CV-MSC CM, and p-STAT3 expression was tested by Immunofluorescence. G Three trophoblast cell lines were cultured under hypoxic conditions with or without cryptotanshinone, IL-6 or CV-MSC CM, and JAK2, p-JAK2, STAT3, p-STAT3, LC3 and P62 expression levels were tested by western blotting analysis. The results are expressed as the means ± SD. ***P < 0.001

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human JAK2, pJAK2, STAT3, pSTAT3, MMP2, MMP9, LC3, BECN1 and P62 (1:1000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL) were incubated with the blocked membranes.

Techniques: Activation Assay, RNA Sequencing, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence

Journal: Cell & Bioscience

Article Title: Chorionic villus-derived mesenchymal stem cell-mediated autophagy promotes the proliferation and invasiveness of trophoblasts under hypoxia by activating the JAK2/STAT3 signalling pathway

doi: 10.1186/s13578-021-00681-7

Figure Lengend Snippet: CV-MSCs promoted trophoblast growth and metastasis through the JAK2/STAT3 signalling pathway. A Cell proliferation was evaluated by a CCK-8 assay. The three trophoblast cell lines were treated with IL-6 or CV-MSC CM and with or without cryptotanshinone under hypoxic conditions. Trophoblast cells cultured alone under hypoxic conditions served as controls. B Trophoblast cells were treated with IL-6 or CV-MSC CM and with or without cryptotanshinone, and the invasion abilities were determined by Transwell assay. The number of penetrated cells was counted and analysed statistically. C The three trophoblast cell lines were cultured under hypoxic conditions with or without 3-MA or CV-MSC CM, and p-JAK2, p-STAT3, MMP2, MMP9 and LC3 expression levels were tested by western blotting analysis. D Cell proliferation was evaluated by a CCK-8 assay. The three trophoblast cell lines were treated with 3-MA or CV-MSC CM under hypoxic conditions. Trophoblast cells cultured alone under hypoxic conditions served as controls. E Trophoblast cells were treated with 3-MA or CV-MSC CM under hypoxic conditions, and invasion abilities were determined by Transwell assay. The number of penetrated cells was counted and analysed statistically. ***P < 0.001

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human JAK2, pJAK2, STAT3, pSTAT3, MMP2, MMP9, LC3, BECN1 and P62 (1:1000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL) were incubated with the blocked membranes.

Techniques: CCK-8 Assay, Cell Culture, Transwell Assay, Expressing, Western Blot

Journal: Cell & Bioscience

Article Title: Chorionic villus-derived mesenchymal stem cell-mediated autophagy promotes the proliferation and invasiveness of trophoblasts under hypoxia by activating the JAK2/STAT3 signalling pathway

doi: 10.1186/s13578-021-00681-7

Figure Lengend Snippet: STAT3 regulates autophagy in trophoblast cells under hypoxic conditions. A Significant increases in STAT3 mRNA were found in JAR, JEG-3 and HTR-8 cells transfected with a STAT3 overexpression plasmid compared to untreated cells (Mock) or empty vector (Vec)-transfected cells under hypoxic conditions by qRT-PCR. B The three trophoblast cell lines were transfected with a STAT3 overexpression plasmid under hypoxic conditions, and MMP2, MMP9, P62 and LC3 expression levels were tested by western blotting analysis. Untreated trophoblast cells (Mock) and empty vector (Vec) cells served as controls. C Cell proliferation was evaluated by a CCK-8 assay. The three trophoblast cell lines were transfected with a STAT3 overexpression plasmid or empty vector under hypoxic conditions. Trophoblast cells cultured alone served as controls. D Trophoblast cells were transfected with a STAT3 overexpression plasmid or empty vector under hypoxic conditions, and invasion abilities were determined by Transwell assay. The number of penetrated cells was counted and analysed statistically. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human JAK2, pJAK2, STAT3, pSTAT3, MMP2, MMP9, LC3, BECN1 and P62 (1:1000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL) were incubated with the blocked membranes.

Techniques: Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Cell Culture, Transwell Assay

Journal: Cell & Bioscience

Article Title: Chorionic villus-derived mesenchymal stem cell-mediated autophagy promotes the proliferation and invasiveness of trophoblasts under hypoxia by activating the JAK2/STAT3 signalling pathway

doi: 10.1186/s13578-021-00681-7

Figure Lengend Snippet: CV-MSC-mediated STAT3 activation increased autophagy in placental explants under hypoxic conditions. A Placental explants were treated with IL-6 or CV-MSC CM and with or without cryptotanshinone under hypoxic conditions, and p-JAK2, p-STAT3, P62 and LC3 expression levels were tested by western blotting analysis. Untreated placental explants under hypoxic conditions served as controls. B Placental explants were treated with IL-6 or CV-MSC CM and with or without cryptotanshinone under hypoxic conditions, and STAT3 and P62 expression levels were tested by immunohistochemistry. Untreated placental explants served as controls (scale bar, 50 μm). C Placental explants were treated with IL-6 or PE-CV-MSC CM and with or without cryptotanshinone under hypoxic conditions, and p-JAK2, p-STAT3, P62 and LC3 expression levels were tested by western blotting. Compared with A , PE-CV-MSC CM showed a lower effect of STAT3 and autophagy activation

Article Snippet: Subsequently, primary rabbit monoclonal antibodies against human JAK2, pJAK2, STAT3, pSTAT3, MMP2, MMP9, LC3, BECN1 and P62 (1:1000 dilution) or β-actin (same dilution; Proteintech, Chicago, IL) were incubated with the blocked membranes.

Techniques: Activation Assay, Expressing, Western Blot, Immunohistochemistry