rabbit anti rubcn  (Proteintech)

Journal: Virulence

Article Title: MAB_0676c-induced enhanced IL-10 production inhibits the autophagic flux via the MTOR/RUBCN pathway

doi: 10.1080/21505594.2025.2529493

Figure Lengend Snippet: M.Abs UC22 inhibits autophagic flux by enhancing RUBCN expression. (a) The number of intracellular bacteria in macrophages. Different cell lines (THP-1, RAW 264.7, and BMDM) were infected with different NTMs. Intracellular bacteria were measured using lysates of infected cells at the indicated times. (b) Western blotting of autophagy-related proteins obtained from the M.Abs-infected BMDMs. Target protein levels were normalized to those of β-actin and are presented as the mean ± SEM from three independent experiments. (c) BMDMs were infected with M.Abs strains for 24 h. Infected cells were stained and visualized using confocal microscopy to detect bacteria (blue), LC3 (green), LAMP2 (red), and DAPI (nuclei; white gray). Scale bars, 2 μm. (d) Quantification of LC3-LAMP2 co-localization based on the images shown in (c). Statistical significance was determined using the one-tailed unpaired t -test. Data are shown as mean ± SEM from three independent experiments. Statistically significant differences are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. ns: not significant. M.Abs, Mycobacterium abscessus subsp. abscessus ; BMDM, bone marrow-derived macrophage; NTM, non-tuberculous mycobacteria; DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: For siRNA knockdown, BMDMs and RAW 264.7 cells were transfected with mouse Rubcn siRNA (Bioneer) or a negative control (Bioneer) using Lipofectamine 3000 (Invitrogen) at an siRNA: Lipofectamine reagent ratio of 1:1.5 according to the manufacturer’s instructions.

Techniques: Expressing, Bacteria, Infection, Western Blot, Staining, Confocal Microscopy, One-tailed Test, Derivative Assay

Journal: Virulence

Article Title: MAB_0676c-induced enhanced IL-10 production inhibits the autophagic flux via the MTOR/RUBCN pathway

doi: 10.1080/21505594.2025.2529493

Figure Lengend Snippet: Knockdown of Rubcn enhances autophagosomal maturation in M.Abs UC22-infected BMDMs. BMDMs were transfected with either the negative control or Rubcn siRNA for 24 h and then infected with M.Abs UC22 at an MOI of 1. (a) Intracellular survival of M.Abs UC22 in transfected BMDMs or RAW 264.7 cells. Data are shown as mean ± SEM from three independent experiments. (b) Western blotting of the autophagy-related proteins in Rubcn knockdown-BMDMs at the indicated time. Target protein levels were normalized to those of β-actin and are presented as mean ± SEM of three independent experiments. (c) si Rubcn - or negative control-transfected BMDMs were infected with M.Abs UC22 at an MOI of 1. Infected cells were stained and visualized under a confocal microscope to detect bacteria (blue), LC3 (green), LAMP2 (red), and DAPI (nuclei; white gray). Scale bars, 2 μm. (d) Quantification of LC3-LAMP2 co-localization based on the images shown in (c). Data are shown as mean ± SEM from three independent experiments. Statistically significant differences are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. ns: not significant. MOI, multiplicity of infection; M.Abs, Mycobacterium abscessus subsp. abscessus ; BMDM, bone marrow-derived macrophage; NC, siRNA negative control; DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: For siRNA knockdown, BMDMs and RAW 264.7 cells were transfected with mouse Rubcn siRNA (Bioneer) or a negative control (Bioneer) using Lipofectamine 3000 (Invitrogen) at an siRNA: Lipofectamine reagent ratio of 1:1.5 according to the manufacturer’s instructions.

Techniques: Knockdown, Infection, Transfection, Negative Control, Western Blot, Staining, Microscopy, Bacteria, Derivative Assay

Journal: Virulence

Article Title: MAB_0676c-induced enhanced IL-10 production inhibits the autophagic flux via the MTOR/RUBCN pathway

doi: 10.1080/21505594.2025.2529493

Figure Lengend Snippet: Identification of MAB_0676c as an inducer of RUBCN expression. (a) Western blot showing RUBCN expression in BMDMs treated with recombinant MAB_0676c and MAB_4702 (5 μg/mL). (b) Western blot showing autophagy-related protein expression in BMDMs treated with recombinant MAB_0676c. BMDMs were incubated with recombinant MAB_0676c protein, heat-inactivated MAB_0676c protein, or LPS for 24 h. (c) Cytotoxicity of MAB_0676c. BMDMs were incubated with recombinant MAB_0676c, STS, or LPS for 24 h. Data are shown as mean ± SEM from three independent experiments. Statistically significant differences are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. ns: not significant. M.Abs, Mycobacterium abscessus subsp. abscessus ; BMDM, bone marrow-derived macrophage; LPS, lipopolysaccharide; STS, staurosporine.

Article Snippet: For siRNA knockdown, BMDMs and RAW 264.7 cells were transfected with mouse Rubcn siRNA (Bioneer) or a negative control (Bioneer) using Lipofectamine 3000 (Invitrogen) at an siRNA: Lipofectamine reagent ratio of 1:1.5 according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Recombinant, Incubation, Derivative Assay

Journal: Virulence

Article Title: MAB_0676c-induced enhanced IL-10 production inhibits the autophagic flux via the MTOR/RUBCN pathway

doi: 10.1080/21505594.2025.2529493

Figure Lengend Snippet: Rubcn knockdown enhances autophagic flux in BMDMs infected with MAB_0676c-expressing M. smegmatis . BMDMs were transfected with either the negative control or Rubcn siRNA for 24 h. Transfected BMDMs were infected with the recombinant M. smegmatis strains at an MOI of 1. (a) Intracellular survival of MAB_0676c-expressing M. smegmatis in si Rubcn -transfected BMDMs. (b) Western blot showing autophagy-related protein expression at the indicated time points. Target protein levels were normalized to those of β-actin and are presented as mean ± SEM from three independent experiments. (c) Infected cells were stained and visualized under a confocal microscope to detect bacteria (blue), LC3 (green), LAMP2 (red), and DAPI (nuclei; white gray). Scale bars, 2 μm. (d) Quantification of LC3-LAMP2 co-localization based on the images shown in (c). Data are shown as mean ± SEM from three independent experiments. Statistically significant differences are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. ns: not significant. MOI, multiplicity of infection; BMDM, bone marrow-derived macrophage; NTM, non-tuberculous mycobacteria; DAPI, 4',6-diamidino-2-phenylindole; NC, siRNA negative control; Ms_VC, M. smegmatis vector control; Ms_MAB0676c, MAB_0676c-expressing M. smegmatis .

Article Snippet: For siRNA knockdown, BMDMs and RAW 264.7 cells were transfected with mouse Rubcn siRNA (Bioneer) or a negative control (Bioneer) using Lipofectamine 3000 (Invitrogen) at an siRNA: Lipofectamine reagent ratio of 1:1.5 according to the manufacturer’s instructions.

Techniques: Knockdown, Infection, Expressing, Transfection, Negative Control, Recombinant, Western Blot, Staining, Microscopy, Bacteria, Derivative Assay, Plasmid Preparation, Control

Journal: Signal transduction and targeted therapy

Article Title: Targeting PI3K inhibitor resistance in breast cancer with metabolic drugs.

doi: 10.1038/s41392-025-02180-4

Figure Lengend Snippet: Fig. 4 CRISPR/Cas9-engineered autophagy-deficiency in breast cancer cells induces metabolic vulnerability. a–f T47DPar cells were infected with plentiCRISPRv2 vectors targeting FIP200, ATG14, ATG7, and RUBCN (sg-1 and sg-2, two independent sgRNAs per gene). After lentiviral transduction and puromycin selection, cells were single-cell cloned and examined for target gene knockout by Western blot (a). Mock: non- infected cells; EV: empty vector control. b Clonogenic growth of control (mock and EV) and knockout cells treated with DCA or Metformin. Shown are representative images. c, d Real-time live-cell imaging of control (mock and EV) and knockout cells treated with DCA or Metformin. c Mean confluence in % over time ± SD (n = 3). FDR q values. d AUC of proliferation curves relative to untreated. Shown is the mean ± SD (n = 3), one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.05; ****p < 0.0001. e Flow cytometry analysis for apoptosis (sub-G1). Indicated knockout cells were treated with DCA or Metformin for 5 days. Shown are mean ± SD, n = 3, two-way ANOVA with Tukey’s multiple comparisons test. f Western blot of control (mock and EV) and knockout cells treated with 40 mM DCA or 4 mM Metformin for 72 h

Article Snippet: Antibodies: Gaussia Luciferase/GLuc+ (1:1000, #401P, Nanolight), Cypridina Luciferase/CLuc+ (1:100, #IT-000-013, Immune Technology), Firefly Luciferase/FLuc+ (1:500, #3848-500, BioVision), cleaved PARP (Asp214) (1:1000, #9541, Cell Signaling), phosphoULK1 (Ser757) (1:1000, #14202, Cell Signaling), total ULK (R600) (1:1000, #4773, Cell Signaling), p62/SQSTM1 (1:1000, P0067, Sigma), LC3B (LC3-I/II) (1:1000, ab48394, Abcam), phosphop70S6Kinase (Thr389) (1:1000, 108D2 #9234, Cell Signaling), total p70S6Kinase (H9) (1:500, sc-8418, Santa Cruz Biotechnology), phospho-4E-BP1 (Thr37/46) (1:1000, 236B4 #2855, Cell Signaling), total 4E-BP1 (R-113) (1:200, #sc-6936, Santa Cruz Biotechnology) phospho-AMPK (Thr172) (40H9) (1:1000, #2535, Cell Signaling), total AMPK (23A3) (1:1000, #2603, Cell Signaling), phospho-AcetylCoA Carboxylase (Ser79) (1:1000, #3661, Cell Signaling), total Acetyl-CoA Carboxylase (C83B10) (1:1000, #3676, Cell Signaling), ATG7 (D12B11) (1:1000, #8558, Cell Signaling), FIP200 (D10D11) (1:1000, #12436, Cell Signaling), ATG14 (1:1000, #5504, Cell Signaling), RUBCN (D9F7) (1:1000, #8465, Cell Signaling), β-Actin (AC-15) (1:10.000, ab6276, Abcam).

Techniques: CRISPR, Infection, Transduction, Selection, Clone Assay, Gene Knockout, Western Blot, Plasmid Preparation, Control, Knock-Out, Live Cell Imaging, Flow Cytometry