Journal: Intervirology

Article Title: The Regulation of Prototype Foamy Virus 5′Long Terminal Repeats and Internal Promoter by Endogenous Transcription Factors

doi: 10.1159/000517539

Figure Lengend Snippet: Plasmids used in the assays

Article Snippet: Then the proteins were transferred to PVDF membrane and incubated with anti-BAG3 (Bioss, China) and anti-fos (Bioss) polyclonal antibodies, respectively.

Techniques: Plasmid Preparation, Expressing

Journal: Intervirology

Article Title: The Regulation of Prototype Foamy Virus 5′Long Terminal Repeats and Internal Promoter by Endogenous Transcription Factors

doi: 10.1159/000517539

Figure Lengend Snippet: Promoter activities of PFV 5′LTR and IP regulated by AP-1 (a) and BAG3 (b). The plasmids pcDNA-jun and pcDNA-fos were used to express the 2 subunits of the heterodimer AP-1. The plasmid pcDNA-BAG3 was used to express the BAG3 protein. At the bottom of each column, the amount (nanogram) of the plasmids co-transfected in each group was labeled. PFV, prototype foamy virus; LTR, long terminal repeat; IP, internal promoter; TRE, Tas responsive elements; AP-1, activator promoter-1; PMA, phorbol-12-myristate-13-acetate; BAG3, BCL2-associated athanogene 3. Asterisks indicate significant difference.

Article Snippet: Then the proteins were transferred to PVDF membrane and incubated with anti-BAG3 (Bioss, China) and anti-fos (Bioss) polyclonal antibodies, respectively.

Techniques: Plasmid Preparation, Transfection, Labeling

Journal: Carcinogenesis

Article Title: MicroRNA 345, a methylation-sensitive microRNA is involved in cell proliferation and invasion in human colorectal cancer.

doi: 10.1093/carcin/bgr114

Figure Lengend Snippet: Fig. 5. BAG3 was identified to be a target of mir-345. (A) Base pairing comparison between mature mir-345 and the WTor mutant (MUT) putative target site in the 3#-UTR of BAG3 mRNA. The mutant sequence is identical to the WT construct except for mutations highlighted in bold disrupting base pairing at the 5# end of mir-345. (B) Dual luciferase assay of HT29 cells co-transfected with vector containing the WT or mutant (MUT) putative BAG3 3#-UTR target site and pri-mir-345 or mir-345 ASO. The luciferase values of pri-mir-345 transfections were normalized to the average values for the corresponding mir-345 ASO transfections. P , 0.01. (C) Western blots of BAG3 in HT29 cells transfected with pri-mir-345. b-actin was used as a loading control. The density of the western blot bands was normalized to the amount of BAG3 protein as determined by the density of the band obtained by western blot for b-actin. P , 0.05.

Article Snippet: Slides were then incubated overnight at 4 C in a humidified chamber with rabbit polyclonal anti-BAG3 antibody (1:200; Abcam, Cambridge, UK) and subsequently with antirabbit IgG for 20 min.

Techniques: Comparison, Mutagenesis, Sequencing, Construct, Luciferase, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Carcinogenesis

Article Title: MicroRNA 345, a methylation-sensitive microRNA is involved in cell proliferation and invasion in human colorectal cancer.

doi: 10.1093/carcin/bgr114

Figure Lengend Snippet: Fig. 6. Western blots and qRT–PCR results of BAG3 in CRC patients. (A) qRT–PCR of BAG3 in CRC tissues (Ca), para-cancer tissues (P) and adjacent non-tumor mucosal tissues (N) of 31 patients. P , 0.001. (B) Western blots of BAG3 in CRC cases 1 and 8 with glyceraldehyde-3-phosphate dehydrogenase as a loading control (bottom panel). Western blots of BAG3 in all of the 31 CRC cases were shown in supplementary Figure 3, available at Carcinogenesis Online. The density of the western blot bands was normalized to the amount of BAG3 protein as determined by the density of the glyceraldehyde-3-phosphate dehydrogenase band. The three columns in the bar represent the mean density of BAG3 bands in the N, P and Ca groups. The amount of BAG3 protein was significantly higher in the Ca group. (C) Immunocytochemistry analysis of BAG3 expression in CRC case (Ca), its normal surrounding tissue (N) and adenoma (A).

Article Snippet: Slides were then incubated overnight at 4 C in a humidified chamber with rabbit polyclonal anti-BAG3 antibody (1:200; Abcam, Cambridge, UK) and subsequently with antirabbit IgG for 20 min.

Techniques: Western Blot, Quantitative RT-PCR, Control, Immunocytochemistry, Expressing

Journal: Journal of Orofacial Orthopedics

Article Title: Role of chaperone-assisted selective autophagy (CASA) in mechanical stress protection of periodontal ligament cells

doi: 10.1007/s00056-021-00358-3

Figure Lengend Snippet: Genetic profile of periodontal ligament (PDL) cells for chaperone-assisted selective autophagy (CASA) complex-characteristic marker genes under different loading intensities. Investigation of expression and transcriptional changes for BAG3, STUB1, HSPA8, HSPB8 , and SYNPO2 in PDL cells. Cultures were exposed to different tension amounts of 2.5, 5, and 10%. Untreated cells served as control. Analyses were performed via RT-qPCR (Quantitative reverse transcription PCR). The results were standardized to the reference gene 18S. Values represent the mean ± standard error of the mean (SEM; n = 6) of the relative differential gene expression compared to untreated cells (fold of control). All data were statistically analyzed using one sample t‑test. P < 0.05 was considered statistically significant Genprofil von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Markergenen in PDL(Parodontalligament)-Zellen bei unterschiedlichen Belastungsgraden. Untersuchung von Expression und transkriptionellen Veränderungen für BAG3, STUB1, HSPA8, HSPB8 und SYNPO2 in PDL-Zellen. Die Kulturen wurden unterschiedlichen Dehnungsbelastungen (2,5, 5 und 10%) ausgesetzt. Als Kontrolle dienten nichtbehandelte Zellen. Die Analysen wurden mittels RT-qPCR durchgeführt, die Ergebnisse wurden auf das Referenzgen 18S normiert. Die Werte stellen den Mittelwert ± Standardfehler des Mittelwerts (SEM; n = 6) der relativen differenziellen Genexpression im Vergleich zu den nichtbehandelten Zellen („fold of control“) dar. Alle Daten wurden mit einem t‑Test für eine Stichprobe statistisch ausgewertet. Als statistisch signifikant wurde p < 0,05 festgelegt

Article Snippet: Incubation of primary antibodies was performed with anti-human HSPA8 (1:200; GTX101144; GeneTex), anti-human HSPB8 (1:100; orb94695; biorbyt), anti-human BAG3 (1:100; GTX102396; GeneTex), anti-human STUB1 (1:100; GTX109676; GeneTex), anti-human SYNPO2 (1:200; GTX85139; GeneTex), and anti-human Filamin A (1:100; kindly provided by Prof. Höhfeld, Insitute for Cell Biology, University of Bonn, Germany), respectively.

Techniques: Marker, Expressing, Control, Quantitative RT-PCR, Reverse Transcription, Gene Expression

Journal: Journal of Orofacial Orthopedics

Article Title: Role of chaperone-assisted selective autophagy (CASA) in mechanical stress protection of periodontal ligament cells

doi: 10.1007/s00056-021-00358-3

Figure Lengend Snippet: Protein expression of chaperone-assisted selective autophagy (CASA) complex-characteristic molecules in periodontal ligament (PDL) cells at rest and under different loading intensities. Protein expression of BAG3, STUB1, HSPA8, HSPB8, SYNPO2, and Filamin A in PDL cells analyzed via western blot. Cultures were exposed to different amounts of mechanical strain (s) of 2.5, 5, and 10%. The left lanes represent the results from the untreated cells serving as control (C), and the right lanes show the results for the different amounts of strain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 37 kDa) was used as protein loading control and is presented for each band below the molecule of interest. kDa kilodalton. Figures feature representative results from one donor Proteinexpression von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Markergenen in PDL(Parodontalligament)-Zellen in Ruhe und bei unterschiedlichen Belastungsgraden. Proteinexpression von BAG3, STUB1, HSPA8, HSPB8, SYNPO2 und Filamin A in PDL-Zellen, analysiert mittels Western-Blot. Die Kulturen wurden unterschiedlichen mechanischen Belastungen (s) ausgesetzt: 2,5, 5 und 10%. Die linken Bahnen zeigen die Ergebnisse der unbehandelten Zellen, die als Kontrolle (C) dienten, die rechten Bahnen die Ergebnisse bei den unterschiedlichen Belastungsgraden. Glyceraldehyd-3-Phosphat-Dehydrogenase (GAPDH, 37 kDa) wurde als Proteinladekontrolle verwendet und ist für jede Bande unterhalb des interessierenden Moleküls angegeben. kDa Kilodalton. Die Abbildungen zeigen repräsentative Ergebnisse von einem Spender

Article Snippet: Incubation of primary antibodies was performed with anti-human HSPA8 (1:200; GTX101144; GeneTex), anti-human HSPB8 (1:100; orb94695; biorbyt), anti-human BAG3 (1:100; GTX102396; GeneTex), anti-human STUB1 (1:100; GTX109676; GeneTex), anti-human SYNPO2 (1:200; GTX85139; GeneTex), and anti-human Filamin A (1:100; kindly provided by Prof. Höhfeld, Insitute for Cell Biology, University of Bonn, Germany), respectively.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Orofacial Orthopedics

Article Title: Role of chaperone-assisted selective autophagy (CASA) in mechanical stress protection of periodontal ligament cells

doi: 10.1007/s00056-021-00358-3

Figure Lengend Snippet: Staining intensity calculation for chaperone-assisted selective autophagy (CASA) complex-characteristic molecules in the periodontal ligament (PDL) of teeth with and without orthodontic tooth movement (OTM). Immunohistochemical quantification of the staining intensities of mechanically loaded ( Moved ) and control ( Control ) specimens for BAG3, STUB1, HSPA8, HSPB8, SYNPO2, and Filamin A. Three regions of interest (ROI) were measured to obtain the mean values for diaminobenzodine (DAB) staining intensities within an image. Values represent the mean ± standard error of the mean (SEM) of n = 6 specimens per group. All data were statistically analyzed using one way analysis of variance (ANOVA) and Bonferroni correction. P < 0.05 was considered statistically significant Berechnung der Färbeintensität von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Moleküle in PDL(Parodontalligament)-Zellen von Zähnen mit und ohne kieferorthopädische Zahnewegung (OTM). Immunhistochemische Quantifizierung der Färbeintensitäten von mechanisch belasteten Proben ( Moved ) und Kontrollproben ( Control ) für BAG3, STUB1, HSPA8, HSPB8, SYNPO2 und Filamin A. Drei ROI („regions of interest“) innerhalb eines Bildes wurden zur Ermittlung der Mittelwerte für DAB(Diaminobenzodin)-Färbeintensitäten vermessen. Die Werte stellen den Mittelwert ±Standardfehler des Mittelwerts (SEM) von n = 6 Proben pro Gruppe dar. Alle Daten wurden mittels Einwegvarianzanalyse (ANOVA) und Bonferroni-Korrektur statistisch ausgewertet. Als statistisch signifikant wurde p < 0,05 festgelegt

Article Snippet: Incubation of primary antibodies was performed with anti-human HSPA8 (1:200; GTX101144; GeneTex), anti-human HSPB8 (1:100; orb94695; biorbyt), anti-human BAG3 (1:100; GTX102396; GeneTex), anti-human STUB1 (1:100; GTX109676; GeneTex), anti-human SYNPO2 (1:200; GTX85139; GeneTex), and anti-human Filamin A (1:100; kindly provided by Prof. Höhfeld, Insitute for Cell Biology, University of Bonn, Germany), respectively.

Techniques: Staining, Immunohistochemical staining, Control

Journal: Journal of Orofacial Orthopedics

Article Title: Role of chaperone-assisted selective autophagy (CASA) in mechanical stress protection of periodontal ligament cells

doi: 10.1007/s00056-021-00358-3

Figure Lengend Snippet: a – u In vivo verification of chaperone-assisted selective autophagy (CASA) complex-characteristic molecules in the periodontal ligament (PDL) of teeth with or without orthodontic tooth movement (OTM). In vivo protein expression of BAG3 ( a – c ), STUB1 ( d – g ), HSPA8 ( h , i ), HSPB8 ( j – m ), SYNPO2 ( n – q ), and Filamin A ( r – u ) in PDL cells visualized by immunohistochemistry on teeth without OTM extracted due to crowding, and on teeth with OTM that were incorporated in a fixed rapid palatal expansion appliance, followed by extraction. Specifications indicated via arrows and arrowheads are explained in the “Results” section. R Root of the tooth, B Bone. Expression intensities were visualized via brown staining with diaminobenzodine (DAB). Magnification × 20 a – u In-vivo-Nachweis von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Molekülen im Parodontalligament (PDL) von Zähnen mit bzw. ohne kieferorthopädische Zahnbewegung (OTM). In-vivo-Proteinexpression von BAG3 ( a – c ), STUB1 ( d – g ), HSPA8 ( h , i ), HSPB8 ( j – m ), SYNPO2 ( n – q ) und Filamin A ( r – u ) in PDL-Zellen, immunhistochemisch visualisiert an Zähnen ohne OTM, die bei Engstand extrahiert wurden, und an Zähnen mit OTM, die im Rahmen einer schnellen Gaumennahterweiterung in eine festsitzende Apparatur integriert und anschließend extrahiert wurden. Die durch Pfeile und Pfeilspitzen gekennzeichneten Spezifikationen werden im Abschnitt „Ergebnisse“ erläutert. R Zahnwurzel, B Knochen. Expressionsstärken wurden durch Braunfärbung mit Diaminobenzodin (DAB) visualisiert. Vergr. 20:1

Article Snippet: Incubation of primary antibodies was performed with anti-human HSPA8 (1:200; GTX101144; GeneTex), anti-human HSPB8 (1:100; orb94695; biorbyt), anti-human BAG3 (1:100; GTX102396; GeneTex), anti-human STUB1 (1:100; GTX109676; GeneTex), anti-human SYNPO2 (1:200; GTX85139; GeneTex), and anti-human Filamin A (1:100; kindly provided by Prof. Höhfeld, Insitute for Cell Biology, University of Bonn, Germany), respectively.

Techniques: In Vivo, Expressing, Immunohistochemistry, Extraction, Staining

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: Oncotarget

Article Title: Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas

doi: 10.18632/oncotarget.7910

Figure Lengend Snippet: ( A ) Representative immunohistochemical staining against Beclin1 of NAB, pilocytic astrocytoma WHO grade I, diffuse astrocytoma WHO grade II, anaplastic astrocytoma WHO grade III and glioblastoma WHO grade IV. Scale bar: 50 μm. ( B ) Box blots showing Beclin1 scores (frequency × intensity) in 47 pilocytic astrocytomas WHO grade I (min: 0, max: 9, median: 2), 9 diffuse astrocytomas WHO grade II (min: 0, max: 6, median: 1), 34 anaplastic astrocytomas WHO grade III (min: 0, max: 9, median: 2) and 242 glioblastomas WHO grade IV (min: 0, max: 12, median: 2) ( C ) Beclin1 immunoblotting of normal grey and white matter and two astrocytomas of each WHO grade. ( D ) Relative Beclin1 mRNA levels of normal grey and white matter and two astrocytomas of each WHO grade normalized to normal white matter assessed by qPCR. ( E ) Representative immunohistochemical staining against BAG3 of NAB, pilocytic astrocytoma WHO grade I, diffuse astrocytoma WHO grade II, anaplastic astrocytoma WHO grade III and glioblastoma WHO grade IV. Scale bar: 50 μm. ( F ) Box blots showing BAG3 scores (frequency × intensity) in 46 pilocytic astrocytomas WHO grade I (min: 0, max: 12, median: 4), 16 diffuse astrocytomas WHO grade II (min: 0, max: 6, median: 1.5), 34 anaplastic astrocytomas WHO grade III (min: 0, max: 6, median: 2) and 242 glioblastomas WHO grade IV (min: 0, max: 6, median: 1) ( G ) BAG3 immunoblotting of normal grey and white matter and two astrocytomas of each WHO grade. ( H ) Relative BAG3 mRNA levels of normal and white matter and two astrocytomas of each WHO grade normalized to normal white matter assessed by qPCR.

Article Snippet: The following primary antibodies were used: anti-human LC3B (mouse antibody, 0260–100, dilution: 1:100, NanoTools, Teningen, Germany), anti-human p62 (mouse antibody, 610832, dilution: 1:500, BD Transduction Laboratories, Franklin Lakes, NJ, USA), anti-human BAG3 (rabbit antibody, PAB0330 dilution: 1:100, Abnova, Taipei City, Taiwan), anti-human Beclin1 (rabbit antibody, 0260–100, dilution: 1:100, Santa Cruz, Dallas, TX, USA), anti-human LAMP2 (rabbit antibody, ab37024, dilution: 1:200, Abcam, Cambridge, MA, USA), anti-human Cathepsin B (goat antibody, sc-6493, dilution: 1:250, Santa Cruz) and anti-human cleaved caspase 3 (rabbit antibody, dilution: 1:100, Cell Signaling, Cambridge, UK).

Techniques: Immunohistochemical staining, Staining, Western Blot

Journal: Oncotarget

Article Title: Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas

doi: 10.18632/oncotarget.7910

Figure Lengend Snippet: qPCR primers

Article Snippet: The following primary antibodies were used: anti-human LC3B (mouse antibody, 0260–100, dilution: 1:100, NanoTools, Teningen, Germany), anti-human p62 (mouse antibody, 610832, dilution: 1:500, BD Transduction Laboratories, Franklin Lakes, NJ, USA), anti-human BAG3 (rabbit antibody, PAB0330 dilution: 1:100, Abnova, Taipei City, Taiwan), anti-human Beclin1 (rabbit antibody, 0260–100, dilution: 1:100, Santa Cruz, Dallas, TX, USA), anti-human LAMP2 (rabbit antibody, ab37024, dilution: 1:200, Abcam, Cambridge, MA, USA), anti-human Cathepsin B (goat antibody, sc-6493, dilution: 1:250, Santa Cruz) and anti-human cleaved caspase 3 (rabbit antibody, dilution: 1:100, Cell Signaling, Cambridge, UK).

Techniques: Sequencing

Journal: Journal of Orofacial Orthopedics

Article Title: Role of chaperone-assisted selective autophagy (CASA) in mechanical stress protection of periodontal ligament cells

doi: 10.1007/s00056-021-00358-3

Figure Lengend Snippet: Genetic profile of periodontal ligament (PDL) cells for chaperone-assisted selective autophagy (CASA) complex-characteristic marker genes under different loading intensities. Investigation of expression and transcriptional changes for BAG3, STUB1, HSPA8, HSPB8 , and SYNPO2 in PDL cells. Cultures were exposed to different tension amounts of 2.5, 5, and 10%. Untreated cells served as control. Analyses were performed via RT-qPCR (Quantitative reverse transcription PCR). The results were standardized to the reference gene 18S. Values represent the mean ± standard error of the mean (SEM; n = 6) of the relative differential gene expression compared to untreated cells (fold of control). All data were statistically analyzed using one sample t‑test. P < 0.05 was considered statistically significant Genprofil von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Markergenen in PDL(Parodontalligament)-Zellen bei unterschiedlichen Belastungsgraden. Untersuchung von Expression und transkriptionellen Veränderungen für BAG3, STUB1, HSPA8, HSPB8 und SYNPO2 in PDL-Zellen. Die Kulturen wurden unterschiedlichen Dehnungsbelastungen (2,5, 5 und 10%) ausgesetzt. Als Kontrolle dienten nichtbehandelte Zellen. Die Analysen wurden mittels RT-qPCR durchgeführt, die Ergebnisse wurden auf das Referenzgen 18S normiert. Die Werte stellen den Mittelwert ± Standardfehler des Mittelwerts (SEM; n = 6) der relativen differenziellen Genexpression im Vergleich zu den nichtbehandelten Zellen („fold of control“) dar. Alle Daten wurden mit einem t‑Test für eine Stichprobe statistisch ausgewertet. Als statistisch signifikant wurde p < 0,05 festgelegt

Article Snippet: Incubation of primary antibodies was performed with anti-human HSPA8 (1:200; GTX101144; GeneTex), anti-human HSPB8 (1:100; orb94695; biorbyt), anti-human BAG3 (1:100; GTX102396; GeneTex), anti-human STUB1 (1:100; GTX109676; GeneTex), anti-human SYNPO2 (1:200; GTX85139; GeneTex), and anti-human Filamin A (1:100; kindly provided by Prof. Höhfeld, Insitute for Cell Biology, University of Bonn, Germany), respectively.

Techniques: Marker, Expressing, Control, Quantitative RT-PCR, Reverse Transcription, Gene Expression

Journal: Journal of Orofacial Orthopedics

Article Title: Role of chaperone-assisted selective autophagy (CASA) in mechanical stress protection of periodontal ligament cells

doi: 10.1007/s00056-021-00358-3

Figure Lengend Snippet: Protein expression of chaperone-assisted selective autophagy (CASA) complex-characteristic molecules in periodontal ligament (PDL) cells at rest and under different loading intensities. Protein expression of BAG3, STUB1, HSPA8, HSPB8, SYNPO2, and Filamin A in PDL cells analyzed via western blot. Cultures were exposed to different amounts of mechanical strain (s) of 2.5, 5, and 10%. The left lanes represent the results from the untreated cells serving as control (C), and the right lanes show the results for the different amounts of strain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 37 kDa) was used as protein loading control and is presented for each band below the molecule of interest. kDa kilodalton. Figures feature representative results from one donor Proteinexpression von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Markergenen in PDL(Parodontalligament)-Zellen in Ruhe und bei unterschiedlichen Belastungsgraden. Proteinexpression von BAG3, STUB1, HSPA8, HSPB8, SYNPO2 und Filamin A in PDL-Zellen, analysiert mittels Western-Blot. Die Kulturen wurden unterschiedlichen mechanischen Belastungen (s) ausgesetzt: 2,5, 5 und 10%. Die linken Bahnen zeigen die Ergebnisse der unbehandelten Zellen, die als Kontrolle (C) dienten, die rechten Bahnen die Ergebnisse bei den unterschiedlichen Belastungsgraden. Glyceraldehyd-3-Phosphat-Dehydrogenase (GAPDH, 37 kDa) wurde als Proteinladekontrolle verwendet und ist für jede Bande unterhalb des interessierenden Moleküls angegeben. kDa Kilodalton. Die Abbildungen zeigen repräsentative Ergebnisse von einem Spender

Article Snippet: Incubation of primary antibodies was performed with anti-human HSPA8 (1:200; GTX101144; GeneTex), anti-human HSPB8 (1:100; orb94695; biorbyt), anti-human BAG3 (1:100; GTX102396; GeneTex), anti-human STUB1 (1:100; GTX109676; GeneTex), anti-human SYNPO2 (1:200; GTX85139; GeneTex), and anti-human Filamin A (1:100; kindly provided by Prof. Höhfeld, Insitute for Cell Biology, University of Bonn, Germany), respectively.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Orofacial Orthopedics

Article Title: Role of chaperone-assisted selective autophagy (CASA) in mechanical stress protection of periodontal ligament cells

doi: 10.1007/s00056-021-00358-3

Figure Lengend Snippet: Staining intensity calculation for chaperone-assisted selective autophagy (CASA) complex-characteristic molecules in the periodontal ligament (PDL) of teeth with and without orthodontic tooth movement (OTM). Immunohistochemical quantification of the staining intensities of mechanically loaded ( Moved ) and control ( Control ) specimens for BAG3, STUB1, HSPA8, HSPB8, SYNPO2, and Filamin A. Three regions of interest (ROI) were measured to obtain the mean values for diaminobenzodine (DAB) staining intensities within an image. Values represent the mean ± standard error of the mean (SEM) of n = 6 specimens per group. All data were statistically analyzed using one way analysis of variance (ANOVA) and Bonferroni correction. P < 0.05 was considered statistically significant Berechnung der Färbeintensität von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Moleküle in PDL(Parodontalligament)-Zellen von Zähnen mit und ohne kieferorthopädische Zahnewegung (OTM). Immunhistochemische Quantifizierung der Färbeintensitäten von mechanisch belasteten Proben ( Moved ) und Kontrollproben ( Control ) für BAG3, STUB1, HSPA8, HSPB8, SYNPO2 und Filamin A. Drei ROI („regions of interest“) innerhalb eines Bildes wurden zur Ermittlung der Mittelwerte für DAB(Diaminobenzodin)-Färbeintensitäten vermessen. Die Werte stellen den Mittelwert ±Standardfehler des Mittelwerts (SEM) von n = 6 Proben pro Gruppe dar. Alle Daten wurden mittels Einwegvarianzanalyse (ANOVA) und Bonferroni-Korrektur statistisch ausgewertet. Als statistisch signifikant wurde p < 0,05 festgelegt

Article Snippet: Incubation of primary antibodies was performed with anti-human HSPA8 (1:200; GTX101144; GeneTex), anti-human HSPB8 (1:100; orb94695; biorbyt), anti-human BAG3 (1:100; GTX102396; GeneTex), anti-human STUB1 (1:100; GTX109676; GeneTex), anti-human SYNPO2 (1:200; GTX85139; GeneTex), and anti-human Filamin A (1:100; kindly provided by Prof. Höhfeld, Insitute for Cell Biology, University of Bonn, Germany), respectively.

Techniques: Staining, Immunohistochemical staining, Control

Journal: Journal of Orofacial Orthopedics

Article Title: Role of chaperone-assisted selective autophagy (CASA) in mechanical stress protection of periodontal ligament cells

doi: 10.1007/s00056-021-00358-3

Figure Lengend Snippet: a – u In vivo verification of chaperone-assisted selective autophagy (CASA) complex-characteristic molecules in the periodontal ligament (PDL) of teeth with or without orthodontic tooth movement (OTM). In vivo protein expression of BAG3 ( a – c ), STUB1 ( d – g ), HSPA8 ( h , i ), HSPB8 ( j – m ), SYNPO2 ( n – q ), and Filamin A ( r – u ) in PDL cells visualized by immunohistochemistry on teeth without OTM extracted due to crowding, and on teeth with OTM that were incorporated in a fixed rapid palatal expansion appliance, followed by extraction. Specifications indicated via arrows and arrowheads are explained in the “Results” section. R Root of the tooth, B Bone. Expression intensities were visualized via brown staining with diaminobenzodine (DAB). Magnification × 20 a – u In-vivo-Nachweis von für den CASA(„chaperone-assisted selective autophagy“)-Komplex charakteristischen Molekülen im Parodontalligament (PDL) von Zähnen mit bzw. ohne kieferorthopädische Zahnbewegung (OTM). In-vivo-Proteinexpression von BAG3 ( a – c ), STUB1 ( d – g ), HSPA8 ( h , i ), HSPB8 ( j – m ), SYNPO2 ( n – q ) und Filamin A ( r – u ) in PDL-Zellen, immunhistochemisch visualisiert an Zähnen ohne OTM, die bei Engstand extrahiert wurden, und an Zähnen mit OTM, die im Rahmen einer schnellen Gaumennahterweiterung in eine festsitzende Apparatur integriert und anschließend extrahiert wurden. Die durch Pfeile und Pfeilspitzen gekennzeichneten Spezifikationen werden im Abschnitt „Ergebnisse“ erläutert. R Zahnwurzel, B Knochen. Expressionsstärken wurden durch Braunfärbung mit Diaminobenzodin (DAB) visualisiert. Vergr. 20:1

Article Snippet: Incubation of primary antibodies was performed with anti-human HSPA8 (1:200; GTX101144; GeneTex), anti-human HSPB8 (1:100; orb94695; biorbyt), anti-human BAG3 (1:100; GTX102396; GeneTex), anti-human STUB1 (1:100; GTX109676; GeneTex), anti-human SYNPO2 (1:200; GTX85139; GeneTex), and anti-human Filamin A (1:100; kindly provided by Prof. Höhfeld, Insitute for Cell Biology, University of Bonn, Germany), respectively.

Techniques: In Vivo, Expressing, Immunohistochemistry, Extraction, Staining

Journal: Journal of advanced research

Article Title: Calenduloside E suppresses calcium overload by promoting the interaction between L-type calcium channels and Bcl2-associated athanogene 3 to alleviate myocardial ischemia/reperfusion injury.

doi: 10.1016/j.jare.2020.10.005

Figure Lengend Snippet: Fig. 5. CE promoted interaction between LTCC and BAG3. (a) DARTS assay is used to detect the effect of CE on BAG3 or a1C. Lysate of NRVMs was hydrolyzed by pronase after CE (0, 100, 200, 400, 800 lM) incubation 1 h. The expression of BAG3 and a1C were showed through western blots and statistical histograms (n = 5 per group). The data were expressed as the mean ± SD. ###P < 0. 001 vs control group; *P < 0.05 vs pronase group, **P < 0.01 vs pronase group, ***P < 0.001 vs pronase group. (b-c) Lysates (sham, CE, IR, CE + IR group) of rat heart tissue were co-immunoprecipitated using BAG3 or a1C antibody. The expression of BAG3 and a1C in input and IP were showed through western blots and statistical histograms (n = 5 per group). The data were expressed as the mean ± SD. ###P < 0. 001 vs sham group; **P < 0.01 vs IR group, ***P < 0.001 vs IR group. (d) Colocalization of BAG3 and a1C by immunofluorescence double staining were showed by the images (The scale bar is 100 lm or 20 lm.) and statistical histograms (n = 5 per group). The data were expressed as the mean ± SD. ###P < 0. 001 vs sham group; **P < 0.01 vs IR group, ***P < 0.001 vs IR group; &&P < 0.01 vs IR + CE group, &&&P < 0.001 vs IR + CE group.

Article Snippet: The primary antibodies in this article were used including: a1C (ab84814, 1:1000) were purchased from abcam (Cambridge, UK); BAG3 (A14826, 1:500) were purchased from ABclonal (Wuhan, China).

Techniques: Incubation, Expressing, Western Blot, Control, Immunoprecipitation, Double Staining

Journal: Journal of advanced research

Article Title: Calenduloside E suppresses calcium overload by promoting the interaction between L-type calcium channels and Bcl2-associated athanogene 3 to alleviate myocardial ischemia/reperfusion injury.

doi: 10.1016/j.jare.2020.10.005

Figure Lengend Snippet: Fig. 6. Regulation of LTCC agonist on BAG3 and calcium pathway proteins. (a) The expression levels of a1C, a2d, BAG3, and cardiac troponin I (CTnI) in rat myocardial tissue were displayed in immunohistochemical images and statistical histograms (n = 5 per group). (b) The expression levels of LTCC subunits (a1C, a2d, c, b) and BAG3 in NRVMs were displayed in western blots and statistical histograms (n = 5 per group). (c) The expression levels of Calmodulin, SERCA, CaMKⅡand p-CaMKⅡ(T286) in NRVMs were displayed in immunohistochemical images and statistical histograms (n = 5 per group). The data were expressed as the mean ± SD. ##P < 0. 01 vs sham (control) group, ###P < 0. 001 vs sham (control) group; *P < 0.05 vs IR (HR) group, **P < 0.01 vs IR (HR) group, ***P < 0.001 vs IR (HR) group; &P < 0.05 vs IR + CE (HR + CE) group, &&P < 0.01 vs IR + CE (HR + CE) group, &&&P < 0.001 vs IR + CE (HR + CE) group.

Article Snippet: The primary antibodies in this article were used including: a1C (ab84814, 1:1000) were purchased from abcam (Cambridge, UK); BAG3 (A14826, 1:500) were purchased from ABclonal (Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Western Blot, Control