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

Journal: Structure (London, England : 1993)

Article Title: Structural basis of pseudoGTPase-mediated protein-protein interactions

doi: 10.1016/j.str.2025.07.009

Figure Lengend Snippet: (A) Cartoon and surface representation of AAGAB psGD:AP1σ3 complex structure with psGD in gold and AP1σ3 in violet. N- and C- termini, relevant secondary structures, and structural elements of both proteins are labeled. Key interfacial residues on psGD are shown as sticks. In the right panel, the complex is rotated by 70° and AP1σ3 is shown as electrostatic surface. Unmodelled residues with poor electron densities are represented by the dashed line. (B) Zoomed-in view of the psGD D151/F153/E155 interaction with AP1σ3. (C) Zoomed-in view of psGD A168 and K52/Y53 interactions with AP1σ3. (D) Superposition of AAGAB psGD:AP1σ3 with the AP1σ:pSTING dileucine peptide complex (PDB: 7R4H). The AP1σ is colored gray and the pSTING dileucine motif peptide is shown as stick in pale yellow. The two leucine residues, L364 and L365 occupying the L(0) and L(+1) positions, are labeled. F153 in AAGAB psGD and V98 in AP1σ3 are shown as sticks and colored orange and violet, respectively. (E) Superposition of AAGAB psGD:AP1σ3 with the AP2core in complex with the dileucine motif cargo peptide (PDB: 6QH6). The AP2σ is colored gray and the dileucine motif peptide is shown as stick in pale green. The two leucine residues, L8 and L9 occupying L(0) and L(+1) positions, are labeled. (F) Superposition of AAGAB psGD:AP1σ3 with AP1 core in closed form (PDB: 1W63). The AP1β and AP1μm subunits are shown as surface and colored blue and yellow, respectively. (G) Superposition of psGD:AP1σ3 with HRas:RasGAP structure (PDB: 1WQ1). HRas and RasGAP are colored gray and greencyan, respectively. The switch I and II regions of HRas are highlighted in red. (H) GST pull-down gel of GST-AP1σ3 co-expressed with His 6 -SUMO tagged WT or mutant AAGAB psGD. AR: A168R; 2YR: Y53R/Y54R; DEF3R: D151R/F153R/E155R. Ulp1 protease treatment is used to confirm the identity of His 6 -SUMO-AAGAB psGD WT . (I) GST pull-down gel of GST-AP2σ1 co-expressed with His 6 -SUMO tagged WT or mutant AAGAB psGD as in (H). Note that the molecular weights of GST-AP2σ1 and His 6 -SUMO-AAGAB psGD are too close for separation on SDS-PAGE. See also and .

Article Snippet: Primary antibody used in this work included monoclonal anti-FLAG antibodies (Millipore-Sigma, #F1804) at a final concentration of 1 μg/mL, polyclonal anti-AP1γ antibodies (Bethyl, #A304–771A) at a final concentration of 1 μg/mL, polyclonal anti-AP2α antibodies (BD Biosciences, #610502) at a final concentration of 1 μg/mL, polyclonal anti-AP1σ antibodies (Bethyl, #A305–396A) at a final concentration of 1 μg/mL, monoclonal anti-AP2σ antibodies (Abcam, #ab92380) at a final concentration of 1 μg/mL, and monoclonal anti-α-tubulin antibodies (DSHB, #12G10) at a final concentration of 43 ng/mL.

Techniques: Labeling, Mutagenesis, SDS Page

Journal: Structure (London, England : 1993)

Article Title: Structural basis of pseudoGTPase-mediated protein-protein interactions

doi: 10.1016/j.str.2025.07.009

Figure Lengend Snippet: (A) Diagrams of HA-tagged full-length AP1 σ subunit and 3xFLAG-tagged WT or mutant AAGAB used in immunoprecipitation (IP) experiments. (B) Representative immunoblots showing the interaction of 3xFLAG-tagged WT or mutant AAGAB with HA-tagged AP1 σ subunit. The 3xFLAG-AAGAB proteins were transiently expressed in AAGAB KO HeLa cells with an empty vector (control) or plasmids encoding the HA-tagged AP1 σ subunit. The 3xFLAG-AAGAB proteins were immunoprecipitated from cell lysates using anti-FLAG antibodies, and the presence of 3xFLAG-AAGAB and HA-tagged AP1 σ subunit in the immunoprecipitants was detected using anti-FLAG and anti-HA antibodies, respectively. (C) Representative confocal microscopy images from three experiments showing surface levels of TfR in AAGAB KO HeLa cells expressing WT or mutant AAGAB. Surface TfR levels of non-permeabilized cells were labeled using anti-TfR antibodies and Alexa Fluor 568-conjugated secondary antibodies (red). Nuclei were stained with Hoechst 33342 (blue). Scale bars: 20 μm. (D) Flow cytometry measurements of the surface levels of TfR in AAGAB KO HeLa cells expressing the indicated proteins. Data are presented as mean ± s.d., n = 3. *** p < 0.001. (E) Representative immunoblots showing the expression of the indicated proteins in AAGAB KO HeLa cell expressing WT or mutant AAGAB. (F) The relative expression levels of indicated proteins were normalized to WT AAGAB samples. Data are presented as mean ± s.d., n = 3. *** p < 0.001, ** p < 0.01, * p < 0.05, n.s., p > 0.05. In (D) and (F), p values were calculated using one-way ANOVA and Dunnett’s multiple comparison test. Mock: cells transfected with an empty vector. See also .

Article Snippet: Primary antibody used in this work included monoclonal anti-FLAG antibodies (Millipore-Sigma, #F1804) at a final concentration of 1 μg/mL, polyclonal anti-AP1γ antibodies (Bethyl, #A304–771A) at a final concentration of 1 μg/mL, polyclonal anti-AP2α antibodies (BD Biosciences, #610502) at a final concentration of 1 μg/mL, polyclonal anti-AP1σ antibodies (Bethyl, #A305–396A) at a final concentration of 1 μg/mL, monoclonal anti-AP2σ antibodies (Abcam, #ab92380) at a final concentration of 1 μg/mL, and monoclonal anti-α-tubulin antibodies (DSHB, #12G10) at a final concentration of 43 ng/mL.

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Plasmid Preparation, Control, Confocal Microscopy, Expressing, Labeling, Staining, Flow Cytometry, Comparison, Transfection

Journal: Poultry Science

Article Title: OTUD1 regulates cytokine expression and related pathways in goose fatty liver by promoting deubiquitination of its target proteins

doi: 10.1016/j.psj.2024.104382

Figure Lengend Snippet: Analysis on the interaction between OTUD1 and AP1G1 proteins as well as AP1G1 ubiquitination in goose fatty and normal livers. (A) Detection of interaction between OTUD1 and AP1G1 in goose liver samples. (B) Detection of AP1G1 ubiquitination and interaction between OTUD1 and AP1G1 proteins in goose fatty and normal livers. β-Tubulin or GAPDH were used as the internal reference proteins. WCL denotes the whole cell lysate, IP denotes immunoprecipitation, IB denotes immunoblotting. The OTUD1 bands are the strongest ones in the immunoblotting images.

Article Snippet: The primary antibodies include OTUD1 (ab122481, abcam, UK), GAPDH (BSM-33033M, Bioss Biotechnology, China), α-Tubulin (2144, Cell Signaling Technology, USA), β-Tubulin (10094-1-AP, Proteintech, China), ACTB (Cat. 81115-1-RR, Proteintech, China), AP1G1 (sc-398867, Santa Cruz Biotechnology, USA), UB (sc-8017, Santa Cruz Biotechnology, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot

Journal: Poultry Science

Article Title: OTUD1 regulates cytokine expression and related pathways in goose fatty liver by promoting deubiquitination of its target proteins

doi: 10.1016/j.psj.2024.104382

Figure Lengend Snippet: Effect of overfeeding on the mRNA and protein expression of AP1G1 in goose liver. (A) The mRNA expression level of AP1G1 in goose fatty liver versus the normal liver (control). ACTB was used as the internal reference gene, n = 6. (B) The image of immunoblots (left) and quantification (right) of AP1G1 protein in goose fatty and normal livers. β-Tubulin was used as the internal reference gene, n = 6. *** denotes P < 0.001 vs. the control group.

Article Snippet: The primary antibodies include OTUD1 (ab122481, abcam, UK), GAPDH (BSM-33033M, Bioss Biotechnology, China), α-Tubulin (2144, Cell Signaling Technology, USA), β-Tubulin (10094-1-AP, Proteintech, China), ACTB (Cat. 81115-1-RR, Proteintech, China), AP1G1 (sc-398867, Santa Cruz Biotechnology, USA), UB (sc-8017, Santa Cruz Biotechnology, USA).

Techniques: Expressing, Control, Western Blot