rab9  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: ER-to-Golgi Trafficking is a Nutrient-Sensitive Checkpoint Linking Glucose Starvation to Cell Surface Remodeling

doi: 10.1101/2025.10.31.685804

Figure Lengend Snippet: (a–f) U2OS cells (wild-type [WT], ULK1/2 double knockout [DKO], ATG7 knockout [KO], or RAB9 KO were transfected with the E-cadherin RUSH construct. After 48 h, cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM) with or without glucose for 1 h, followed by biotin treatment for 30 min. ( a ) Representative immunofluorescence images captured following anti-GM130 and DAPI staining show changes in reporter distribution after biotin addition and/or glucose starvation. ( b ) Percentage of cells (mean ± S.E.M., n = 3 biological replicates, >37 cells per replicate) with Golgi-localized E-cadherin. ( c–f ) Representative immunoblot analyses show protein levels of ULK1, ULK2, ATG7, or RAB9 in WT and indicated KO cell lines; * indicates nonspecific band in ( e ). ( g–i ) Rescue of glucose starvation–induced ER-to-Golgi trafficking inhibition was evaluated in U2OS ULK1/2 DKO cells stably expressing empty vector (EV), WT ULK1, kinase-dead ULK1 (K46A), or AMPK-resistant ULK1 (4SA). Cells were transfected with the E-cadherin RUSH construct, incubated in DMEM with or without glucose for 1 h, and treated with biotin for 30 min. ( g ) Representative immunofluorescence images captured following anti-GM130 and DAPI staining show changes in reporter distribution after biotin addition and/or glucose starvation. ( h ) Golgi-localized E-cadherin in U2OS ULK1/2 DKO cells expressing WT or mutant forms of ULK (mean ± S.E.M., n = 3 biological replicates, >30 cells per replicate). ( i ) Representative immunoblot analysis shows AMPK activity as indicated by total and phospho-ACC, and ULK1 expression, and AMPK-mediated phosphorylation of ULK1 at S555 in the indicated cell lines. ( j–l ) WT U2OS cells were transfected with the E-cadherin RUSH construct. After 48 h, cells were pre-treated with compound C (5 μM, 4 h) or MRT68921 (1 μM, 24 h) and incubated in DMEM with or without glucose for 1 h, followed by biotin treatment for 30 min. ( j ) Representative immunofluorescence images following anti-GM130 and DAPI staining show changes in reporter distribution after biotin addition and/or glucose starvation. ( k ) Golgi-localized E-cadherin in U2OS cells before and after glucose starvation (mean ± S.E.M., n = 3 biological replicates, >103 cells per replicate). ( l ) Representative immunoblot analysis demonstrates activity of each inhibitor post biotin treatment. ( m–o ) WT or Prkaa1/2 (AMPKα1/2) DKO murine embryonic fibroblasts (MEFs) were transfected with the E-cadherin RUSH construct. After 48 h, cells were incubated in DMEM with or without glucose for 1 h, followed by biotin treatment for 30 min. ( m ) Representative immunofluorescence images captured following anti-GM130 and DAPI staining show changes in reporter distribution after biotin addition and/or glucose starvation. ( n ) Golgi-localized E-cadherin in in WT and AMPK α1/2 DKO MEFs before and after glucose starvation (mean ± S.E.M., n = 3 biological replicates, >33 cells per replicate). ( o ) Levels of phosphorylated ACC and ULK1 in WT and AMPK α1/2 DKO MEFs after 1-h glucose starvation. Statistical significance was analyzed with one-way ANOVA followed by Tukey’s multiple comparisons. ***P < 0.001, **P < 0.01, *P < 0.05; ns, not significant. Scale bar, 10 μm.

Article Snippet: After blocking the membrane with 5% skim milk, blots were probed with antibodies directed against the following targets: ULK1 (Cat. No. 8058, Cell Signaling), p-ULK1 (S555) (Cat. No. 5869, Cell Signaling), ATG7 (Cat. No. 2631, Cell Signaling), ULK2 (Cat. No. HPA009027, Sigma-Aldrich), ATG13 (Cat. No. SAB4200100, Millipore Sigma), p-ATG13 (S318) (Cat. No. 600-401-C49 Rockland), GFP (Cat. No. ab6556, Abcam), FLAG (Cat. No. F1804, Millipore Sigma), HA (Cat. No. 3724, Cell Signaling), GAPDH (Cat. No. G9545, Sigma Aldrich), p-ACC (S79) (Cat. No. 11818, Cell Signaling), ACC (Cat. No. 3676, Cell Signaling), p-S6 (S235/236) (Cat. No. 4858, Cell Signaling), S6 (Cat. No. 2317, Cell Signaling), SEC24C (Cat. No. 8531, Cell Signaling), SEC24A (Cat. No. 15958-1-AP, Proteintech), SEC24B (Cat. No. A304-876A, Bethyl), SEC24D (Cat. No. 14687, Cell Signaling), and RAB9 (Cat. No. 5133, Cell Signaling).

Techniques: Double Knockout, Knock-Out, Transfection, Construct, Incubation, Modification, Immunofluorescence, Staining, Western Blot, Inhibition, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Activity Assay, Phospho-proteomics