Journal: Science Advances

Article Title: Bi-handed assembly chaperones regulate protein complex assembly through an intramolecular handover mechanism

doi: 10.1126/sciadv.adw9158

Figure Lengend Snippet: ( A ) Diagram of pull-down assays detecting AAGAB binding to α and σ2. His 6 -SUMO–tagged WT AAGAB was coexpressed with GST-tagged α (trunk domain, amino acids 1 to 621), untagged WT σ2, or both in E. coli . Lysates from E. coli expressing the indicated proteins were used for pull-down assays. ( B ) Representative Coomassie blue–stained gels (top) and immunoblots (bottom) showing the binding of His 6 -SUMO–tagged WT AAGAB to GST-tagged α, untagged σ2, or both, as depicted in (A). ( C ) Representative immunoblots showing the interaction of 3xFLAG-tagged AAGAB with HA-tagged α and σ2 in AAGAB KO HeLa cells. AAGAB was immunoprecipitated using anti-FLAG antibodies, and proteins in the immunoprecipitates were detected via immunoblotting. ( D ) AlphaFold-predicted structural models of the AAGAB-CTD:α dimer and the AAGAB-CTD:α:σ2 trimer. The dimer prediction was performed using the CTD (amino acids 258 to 315) of AAGAB and the α trunk domain, whereas the trimer prediction was performed using the CTD of AAGAB, the α trunk domain, and WT σ2. The PDB/CIF files of these structural models are included in datasets S1 and S3. ( E ) Diagram of a GST pull-down assay detecting the binding of AAGAB (WT, GD, or CTD) to α and σ2. ( F ) Representative Coomassie blue–stained gels showing the binding of AAGAB to GST-tagged α and untagged σ2 as depicted in (E). ( G and H ) Quantification of GST-tagged α and untagged σ2 based on Coomassie blue–stained gels from four independent experiments, including data shown in (F). Data are presented as mean ± SD. *** P < 0.001; n.s., P > 0.05 (calculated using one-way ANOVA with Holm-Sidak corrections). ( I ) Representative Coomassie blue–stained gels showing His 6 -SUMO–tagged AAGAB isolated using nickel beads from the samples described in (E). ( J ) Quantification of His 6 -SUMO–tagged AAGAB based on Coomassie blue–stained gels.

Article Snippet: The endogenous AP2 α subunit was labeled using anti–α-adaptin antibodies (Santa Cruz Biotechnology, #sc-17771, RRID: AB_2274034) and Alexa Fluor 568–conjugated secondary antibodies (Thermo Fisher Scientific, #A11004, RRID: AB_2534072) or Alexa Fluor 488–conjugated secondary antibodies (Thermo Fisher Scientific, #A11008, RRID: AB_142165).

Techniques: Binding Assay, Expressing, Staining, Western Blot, Immunoprecipitation, Pull Down Assay, Isolation

Journal: Journal of Biological Chemistry

Article Title: RLIP, an Effector of the Ral GTPases, Is a Platform for Cdk1 to Phosphorylate Epsin during the Switch Off of Endocytosis in Mitosis

doi: 10.1074/jbc.m302191200

Figure Lengend Snippet: FIG. 4. RLIP76 is complexed in vivo with AP2 and Numb throughout the cell cycle. A, exponentially growing HeLa cells were transfected with siRNA or mock transfected. 48 h later, cell extracts were solubilized in 1% Triton buffer, and 200 g was submitted to immunoprecipitation with anti-RLIP76 antibodies (Jullien-Flores et al. (9)). After SDS-PAGE, immunoprecipitates were tested by Western blot and immunodetection with antibodies against RalBP1, Numb, and the heavy chain of the adaptin AP2. B, cell extracts from Schneider cells (see Fig. 2C) were immunoprecipitated with serum raised against fly RLIP or with pre-immune serum (pi) and were probed with anti-Numb antibodies (Transduction Laboratories). C, cell extracts from exponen- tially growing HeLa cells (E) and nocodazole-treated cells (M) were solubilized in 1% Triton buffer, and 200 g was submitted to immuno- precipitation with anti-RLIP76 antibodies (9). After SDS-PAGE, immu- noprecipitates were tested by Western blot and immunodetection with antibodies against RLIP76, Numb, the subunit of AP2, Epsin, and cyclinB.

Article Snippet: Monoclonal antibodies to cyclinB1, Numb, and the alpha subunit of adaptin AP2 were purchased from Transduction Laboratories, and a goat polyclonal serum to Epsin1 was purchased from Santa Cruz Biotechnologies.

Techniques: In Vivo, Transfection, Immunoprecipitation, SDS Page, Western Blot, Immunodetection, Transduction

Journal: Journal of Biological Chemistry

Article Title: RLIP, an Effector of the Ral GTPases, Is a Platform for Cdk1 to Phosphorylate Epsin during the Switch Off of Endocytosis in Mitosis

doi: 10.1074/jbc.m302191200

Figure Lengend Snippet: FIG. 6. Epsin forms a cell cycle-independent complex with AP2. Protein extracts from mitotic cells (M) and from exponentially growing cells (E) were immunoprecipitated with anti-Epsin antibodies, and immunoprecipitates were probed for the presence of Epsin and AP2 (alpha chain).

Article Snippet: Monoclonal antibodies to cyclinB1, Numb, and the alpha subunit of adaptin AP2 were purchased from Transduction Laboratories, and a goat polyclonal serum to Epsin1 was purchased from Santa Cruz Biotechnologies.

Techniques: Immunoprecipitation

Journal: Microbiology Spectrum

Article Title: Attenuate host susceptibility to respiratory virus invasion by inhibiting interactions between host proteins SLC16A3 and AP1G1

doi: 10.1128/spectrum.03116-24

Figure Lengend Snippet: The interactions between SLC16A3 and AP1G1 ( A ) TPCA-MS experimental scheme. ( B–D ) SLC16A3 and AP1G1 exhibit similar melting curves. Cells were non-infected or infected with H1N1, RSV, and 229E viruses. ( E ) IP-WB result demonstrating the interaction between SLC16A3 and AP1G1. ( F–H ) Immunofluorescence images illustrating the subcellular localization of SLC16A3 and AP1G1. BEAS2B cells were infected with H1N1, RSV, and 229E viruses. ( I–K ) Western blot assay showing the subcellular localization of AP1G1. In = input, C = cytoplasm, M = cytomembrane. Cells were infected with H1N1, RSV, and 229E virus (MOI = 2, n = 3).

Article Snippet: Primary antibodies of AP1G1 (NBP3-15727) were purchased from Novus Biologicals (Shanghai, China), and SLC16A2 (20676-1-AP) was obtained from Proteintech (Wuhan, China).

Techniques: Infection, Immunofluorescence, Western Blot, Virus

Journal: Microbiology Spectrum

Article Title: Attenuate host susceptibility to respiratory virus invasion by inhibiting interactions between host proteins SLC16A3 and AP1G1

doi: 10.1128/spectrum.03116-24

Figure Lengend Snippet: SFJD intervention with SLC16A3-AP1G1 interaction. ( A ) KEGG Strip Pathway. ( B ) Lactate expression levels in BALF of mice before and after SFJD administration after infection with H1N1 virus. t -test, * P < 0.05, ** P < 0.01, n = 6. ( C ) SLC16A3 expression levels in lung tissue of mice before and after SFJD administration after infection with H1N1 virus ( n = 6). ( D ) IP-WB assay illustrating the attenuated interaction between SLC16A3 and AP1G1 following SFJD treatment ( n = 3). ( E–G ) Subcellular localization of AP1G1 before and after SFJD administration following infection with H1N1, RSV, and 229E viruses. Cells were infected with H1N1, RSV, and 229E virus (MOI = 2, n = 3).

Article Snippet: Primary antibodies of AP1G1 (NBP3-15727) were purchased from Novus Biologicals (Shanghai, China), and SLC16A2 (20676-1-AP) was obtained from Proteintech (Wuhan, China).

Techniques: Stripping Membranes, Expressing, Infection, Virus