Journal: iScience

Article Title: STUB1-mediated ubiquitination regulates the stability of GLUD1 in lung adenocarcinoma

doi: 10.1016/j.isci.2023.107151

Figure Lengend Snippet: STUB1 interacts with GLUD1 and affects the stability of GLUD1 protein (A) Ubibrowser was used to search for E3 ligases of GLUD1. (B and C) In A549 cells, STUB1 was overexpressed (B) or knocked down (C) to detect GLUD1 protein by Western blot. (D) The protein level of GLUD1 and STUB1 in PC9, A549, H292, H358, and H1299 cells were detected by Western blot. (E and F) 293T cells were transfected with pcDNA3.1-His-GLUD1 plasmid (E) or pCMV-Flag-STUB1 (F). The immunoprecipitants were blotted with anti-His or anti-Flag antibodies. WCL: the whole cell lysate. (G and H) In A549 cells, endogenous interaction between GLUD1 and STUB1 was tested using anti-STUB1 (G) and anti-GLUD1 (H) antibodies for co-IP, followed by Western blot detection. (I and J) A549 cells were transfected with Flag-STUB1 plasmid (I) or STUB1 siRNAs (J), the mRNA expression of GLUD1 was detected by RT-PCR. Data represent the average of three independent experiments (mean ± SD). ns, p > 0.05 ∗∗∗p < 0.001. (K and L) A549 cells were transfected with Flag-STUB1 (K) or STUB1 siRNAs (L), cells were treated with CHX (20 μg/mL) for 0, 3, 6, and 12 h. The protein stability of GLUD1 was detected by Western blot (left panel). Relative GLUD1 expression over β-actin was quantified (right panel). Data represent the average of three independent experiments (mean ± SD). ns, p > 0.05; ∗∗p < 0.01 ∗∗∗p < 0.001. (M) In A549 cells, Flag-STUB1 and His-GLUD1 plasmids were co-transfected and treated with MG132 for 12 h. The ubiquitination of GLUD1 was detected. (N and O) In 293T cells, Flag-STUB1 and His-GLUD1 plasmids were co-transfected and treated with MG132 or CQ for 12 h. The K48-linkage ubiquitination (N) and K63-linkage ubiquitination (O) of GLUD1 was detected.

Article Snippet: Anti-STUB1 Recombinant Rabbit Monoclonal Antibody , HUABIO , Cat No: ET7108-65.

Techniques: Western Blot, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: iScience

Article Title: STUB1-mediated ubiquitination regulates the stability of GLUD1 in lung adenocarcinoma

doi: 10.1016/j.isci.2023.107151

Figure Lengend Snippet: Lysine 503 of GLUD1 is the key ubiquitination site regulated by STUB1 (A) The ubiquitination sites of GLUD1 predicted by PhosphoSitePlus. (B–M) The GLUD1 lysine residue mutation plasmids (lysine to arginine) are constructed based on the predicted ubiquitin sites of GLUD1. A549 cells were transfected with these plasmids and pCMV-Flag-STUB1, the ubiquitination levels of GLUD1 were detected. (B) GLUD1 K503R (C) GLUD1 K84R (D) GLUD1 K183R (E) GLUD1 K187R (F) GLUD1 K191R (G) GLUD1 K200R (H) GLUD1 K363R (I) GLUD1 K365R (J) GLUD1 K386R (K) GLUD1 K399R (L) GLUD1 K480R (M). In A549 cells, wild-type GLUD1 (His-GLUD1 WT ) or His-GLUD1 K503R plasmids were overexpressed, CHX (20 μg/mL) was added to cells for 0, 3, 6, 9, 12, and 24 h, and the degradation rate of GLUD1 was detected by Western blot (left panel). Relative GLUD1 expression over β-actin was quantified. Data represent the average of three independent experiments (mean ± SD). ∗∗∗p < 0.001 (right panel). (N) A549 cells were transfected with His-GLUD1 K503R alone or co-transfected with Flag-STUB1, cells were treated with CHX (20 μg/mL) for 0, 3, 6, 9, 12, and 24 h. The degradation rate of GLUD1 was detected by Western blot (left panel). Relative GLUD1 expression over β-actin was quantified. Data represent the average of three independent experiments (mean ± SD). ns, p > 0.05 (right panel).

Article Snippet: Anti-STUB1 Recombinant Rabbit Monoclonal Antibody , HUABIO , Cat No: ET7108-65.

Techniques: Ubiquitin Proteomics, Residue, Mutagenesis, Construct, Transfection, Western Blot, Expressing

Journal: iScience

Article Title: STUB1-mediated ubiquitination regulates the stability of GLUD1 in lung adenocarcinoma

doi: 10.1016/j.isci.2023.107151

Figure Lengend Snippet: The regulation of GLUD1 on cell viability relies on STUB1-mediated ubiquitination and degradation (A and B) A549 and H1299 cells were transfected with His-GLUD1 and Flag-STUB1 plasmid or control plasmid. Cell proliferation assays (upper panel) were performed. GLUD1 expression was detected by Western blot (bottom panel). Data represent the average of three independent experiments (mean ± SD). ns, p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (C and D) A549 and H1299 cells were transfected with His-GLUD1 and STUB1 siRNAs. Cell proliferation assay was performed (upper panel). GLUD1 expression was detected by Western blot (bottom panel). Data represent the average of three independent experiments (mean ± SD). ∗p < 0.05, ∗∗p < 0.01. (E) Cell proliferation assay was performed in A549, A549-GLUD1, and A549-GLUD1 K503R cells (left panel). Protein expression was detected by Western blot (right panel). Data represent the average of three independent experiments (mean ± SD). ∗∗∗p < 0.001. (F) Colony formation assay were performed in A549, A549-GLUD1, and A549-GLUD1 K503R cells (left panel). The graph is statistical analysis (right panel). Data represent the average of three independent experiments (mean ± SD). ∗p < 0.05, ∗∗p < 0.01. (G and H) A549-WT, A549-GLUD1, and A549-GLUD1 K503R cells were subcutaneously injected into the flanks of nude mice. After 25 days, tumors were dissected out and photographed (left panel). Tumor weight and volume were measured (right panel). The p value was calculated by paired t test. ∗p < 0.05, ∗∗p < 0.01. (I and J) Immunohistochemical staining of tumors derived from nude mice using anti-Ki67 (I) or anti-TTF1 (J) antibodies. Magnification is ×200.

Article Snippet: Anti-STUB1 Recombinant Rabbit Monoclonal Antibody , HUABIO , Cat No: ET7108-65.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Proliferation Assay, Colony Assay, Injection, Immunohistochemical staining, Staining, Derivative Assay

Journal: iScience

Article Title: STUB1-mediated ubiquitination regulates the stability of GLUD1 in lung adenocarcinoma

doi: 10.1016/j.isci.2023.107151

Figure Lengend Snippet:

Article Snippet: Anti-STUB1 Recombinant Rabbit Monoclonal Antibody , HUABIO , Cat No: ET7108-65.

Techniques: Ubiquitin Proteomics, Recombinant, Virus, Transfection, SYBR Green Assay, Construct, Plasmid Preparation, Software

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