Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication.

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: Fig. 5. pVI of HAdV-B7 interacted with the host protein BAG3. A HEK 293T cells were transfected with plasmids encoding BAG3-HA (2 μg) and pVI-Flag (2 μg) or vector-Flag (2 μg) for 24 h, followed by Co-IP with anti-Flag binding beads and immunoblot analysis with the indicated antibodies. B HEK 293T cells were transfected with plasmids encoding pVI-Flag (2 μg) and BAG3-HA (2 μg) or vector-HA (2 μg) for 24 h, followed by Co-IP with anti-HA binding beads and immunoblot analysis with the indicated antibodies. C A549 cells were transfected with plasmids encoding BAG3-HA (2 μg) and pVI-Flag (2 μg) or vector-Flag (2 μg) for 24 h, followed by Co-IP with anti-Flag binding beads and immunoblot analysis with the indicated antibodies. D A549 cells were transfected with plasmids encoding pVI-Flag (2 μg) and BAG3- HA (2 μg) or vector-HA (2 μg) for 24 h, followed by Co-IP with anti-HA binding beads and immunoblot analysis with the indicated antibodies. E HEK 293T cells were transfected with vector-Flag (2 μg) or pVI-Flag (2 μg). Cell lysates were evaluated by Western blotting using specific antibodies against BAG3 and LC3. Representative immunoblots were showed and densitometric analysis of the relative protein expression were from three independent experiments. *, P < 0.05. F HEK 293T cells were transfected with plasmids encoding pVI-Flag (2 μg) and BAG3-HA (2 μg), followed by labeling Flag, HA and LAMP1 with a specific primary antibody. The cell nucleus were stained with DAPI. Fluorescence signals were observed using confocal immunofluorescence microscopy. G Quantitative analysis of colocalized immunofluo- rescence intensity was using ImageJ, and approximately 25–30 cells in total for each condition were used for quantification. *, P < 0.05. Scale bars: 5 μm.

Article Snippet: Rabbit antiSQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Expressing, Labeling, Staining, Fluorescence, Microscopy

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication.

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: Fig. 6. BAG3 interacted with pVI in a WW domain-dependent manner. A Schematic diagram of BAG3-WT and the truncation mutants BAG3-ΔN and BAG3-ΔC, highlighting the locations of the functional domains, including the N-terminal WW domain (blue) and the C-terminal BAG domain (red). All three proteins contained HA tags. B HEK 293T cells transfected with the indicated plasmid (2 μg) and BAG3-WT or truncation mutant proteins were detected by Western blotting. C Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-Flag binding beads. BAG3-WT or truncation mutant proteins were detected in the precipitates by Western blotting using an anti-HA-specific primary antibody. D Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-HA binding beads. pVI was detected in the precipitates by Western blotting using an anti- Flag-specific primary antibody.

Article Snippet: Rabbit antiSQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Functional Assay, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Immunoprecipitation, Binding Assay

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication.

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: Fig. 7. BAG3 interacted with the PPSY structural domain of pVI through its WW structural domain. A Schematic diagram of pVI-WT, pVI truncation mutants, and PPSY mutants (PAGG). All five proteins contained Flag tags. B HEK 293T cells transfected with the indicated plasmid (2 μg) and pVI-WT or truncation mutant proteins were detected by Western blotting. C Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-Flag binding beads. BAG3-ΔC was detected in the precipitates by Western blotting using an anti-HA-specific primary antibody. D Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-HA binding beads. pVI-WT or mutant proteins were detected in the precipitates by Western blotting using an anti-Flag-specific primary antibody. E Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-Flag binding beads. BAG3-WT or mutant proteins were detected in the precipitates by Western blotting using an anti-HA-specific primary antibody. F Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-HA binding beads. pVI 1–170 was detected in the precipitates by Western blotting using an anti-Flag-specific primary antibody.

Article Snippet: Rabbit antiSQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Immunoprecipitation, Binding Assay

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication.

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: Fig. 8. BAG3-induced autophagy inhibited viral replication. A549 cells (A) and 16HBE cells (B) were transfected with si BAG3 or siNC. Cell lysates were subjected to Western blotting analysis 24 h after transfection. A549 cells (C) and 16HBE cells (D) were transfected with plasmids encoding BAG3-HA or vector-HA for 24 h. Cell lysates were subjected to Western blotting analysis at 24 h after transfection to detect the expression levels of BAG3, p62, LC3, DBP by using specific antibodies. Representative immunoblots were showed and densitometric analysis of the relative protein expression were from three independent experiments. Statistical sig- nificance was analyzed by Student's t-test, *, P < 0.05; **, P < 0.01. A549 cells (E) and 16HBE cells (F) were transfected with si BAG3 or siNC. Cell lysates were subjected to qPCR analysis using E1A-specific primers. A549 cells (G) and 16HBE cells (H) were transfected with plasmids encoding BAG3-HA or vector-HA for 24 h. Cell lysates were subjected to qPCR analysis using E1A-specific primers. Quantification of the relative mRNA levels of E1A from three independent experiments. Statistical significance was analyzed by Student's t-test, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Rabbit antiSQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Transfection, Western Blot, Plasmid Preparation, Expressing

Journal: Acta Neuropathologica

Article Title: A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8

doi: 10.1007/s00401-017-1756-0

Figure Lengend Snippet: Aggregates of myofibrillar components and autophagy defects in the muscle of Hspb8 K141N/K141N mice. a – c αB-crystallin immunohistochemistry in 12-month-old Hspb8 +/+ ( a ), Hspb8 K141N/K141N ( b ) and Hspb8 −/− ( c ) mice, with notable dense subsarcolemmal and intermyofibrillar aggregates in Hspb8 K141N/K141N mice ( b ). d , f Desmin immunohistochemistry in 12-month-old Hspb8 +/+ ( d ), Hspb8 K141N/K141N ( e ) and Hspb8 −/− ( f ) mice showing aggregates (asterisk) in some myofibers of Hspb8 K141N/K141N mice ( e ). g , h Hspb8 immunohistochemistry in 12-month-old Hspb8 +/+ ( g ), in 12-month-old Hspb8 K141N/K141N , showing numerous myofibers containing aggregates ( h ), and in 12-month-old Hspb8 −/− ( i ). j Western blot showing Hspb8, Bag3, p62, and LC3B in muscle lysates of 2-and 12-month-old Hspb8 K141N/K141N and WT mice. GAPDH is used as a loading control. k Quantification of the Hspb8, Bag3, p62, and LC3BII bands normalized on GAPDH. * p < 0.05. Values are expressed as mean ± SD, N = 3

Article Snippet: The following antibodies were used: Hsp22 (3059, Cell Signaling, Danvers, MA, USA), N-terminal Hsp22 (SAB2101100, Sigma-Aldrich, St Louis, MO, USA), BAG3 (A302-806A-M, Bethyl Laboratories, Montgomery, TX, USA), LC3B (L7543, Sigma-Aldrich, St Louis, MO, USA), p62 (sc-25575, Santa Cruz Biotechnology Inc., Dallas, TX, USA), GAPDH (GTX627408, Gene Tex Inc., Irvine, CA, USA), and the following HRP-conjugated secondary antibodies: anti-mouse IgG1 (1070-05, Southern Biotec, Birmingham, AL, USA), anti-mouse IgG2b (1090-05, Southern Biotec, Birmingham, AL, USA), and anti-rabbit (W401B, Promega, Madison, WI, USA).

Techniques: Immunohistochemistry, Western Blot, Control

Journal: Acta Neuropathologica

Article Title: A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8

doi: 10.1007/s00401-017-1756-0

Figure Lengend Snippet: Hspb8 aggregates and low autophagy power in the sciatic nerve of Hspb8 K141N/K141N mice. a , b Hspb8 immunohistochemistry in 18-month-old WT ( a ) and Hspb8 K141N/K141N ( b ) mice showing that only the Hspb8 K141N/K141N mice develop aggregates ( b ). c Western blot showing Hspb8, Bag3 and p62 and in sciatic nerve lysates of 2-and 12-month-old Hspb8 K141N/K141N and WT mice. GAPDH is used as a loading control. d Quantification of the Hspb8, Bag3, and p62 bands normalized on GAPDH. * p < 0.05. Values are expressed as mean ± SD, N = 3

Article Snippet: The following antibodies were used: Hsp22 (3059, Cell Signaling, Danvers, MA, USA), N-terminal Hsp22 (SAB2101100, Sigma-Aldrich, St Louis, MO, USA), BAG3 (A302-806A-M, Bethyl Laboratories, Montgomery, TX, USA), LC3B (L7543, Sigma-Aldrich, St Louis, MO, USA), p62 (sc-25575, Santa Cruz Biotechnology Inc., Dallas, TX, USA), GAPDH (GTX627408, Gene Tex Inc., Irvine, CA, USA), and the following HRP-conjugated secondary antibodies: anti-mouse IgG1 (1070-05, Southern Biotec, Birmingham, AL, USA), anti-mouse IgG2b (1090-05, Southern Biotec, Birmingham, AL, USA), and anti-rabbit (W401B, Promega, Madison, WI, USA).

Techniques: Immunohistochemistry, Western Blot, Control