anti gapdh  (Novus Biologicals)

Journal: Scientific Reports

Article Title: UGT8/GalCer-dependent resistance of breast cancer cells to drug-induced apoptosis is potentially regulated by the LIM/homeobox protein LHX6

doi: 10.1038/s41598-026-42260-1

Figure Lengend Snippet: ( A ) Schematic representation of the UGT8 gene promoter region designated as the UGT8 response element (UGT8RE) as described in the text. ( B ) Binding of nuclear proteins from MDA-MB-231 cells to UGT8 response element (UGT8RE) fragments analyzed by EMSA in the presence (+) and absence (−) of nuclear lysate. EMSA was performed with double-stranded biotin-labeled oligonucleotide probes. ( C ) Transcription factors (TFs) that bind at least twice to UGT8RE. The UGT8RE3A, UGT8RE2B, and UGT8RE1B fragments were subjected to in silico analysis using the JASPAR database. ( D ) Expression of TF mRNAs in BC cell lines MCF7, T47D, and MDA-MB-231. qPCR was used to analyze TF mRNAs. Their expression levels were normalized against GAPDH, and cells with the lowest expression levels of TF were used as a calibrator sample. All values are mean ± SD of at least 2 independent experiments, each assayed in triplicate. *** p < 0.001. ( E ) Western blotting analysis of TFs expression in BC cell lines. Rabbit polyclonal antibodies anti-GSX1, anti-SOX4, and anti-LHX6 were used to detect TFs in nuclear extracts. For Western blotting, 40 μg of proteins were separated by SDS-PAGE under reducing conditions on a 12% gel and electrophoretically transferred to a nitrocellulose membrane. H3 was used as an internal control.

Article Snippet: To detect specific proteins, the membrane slices were incubated with appropriate primary antibodies: anti-UGT8 (Cat. No. orb214717, Biorbyt), anti-GSX1 (Cat. No. BS-11612R, Thermo), anti-SOX4 (Cat. No. PA595290 , Thermo), anti-LHX6 (Cat. No. orb1535192, Biorbyt) and mouse monoclonal anti-GAPDH antibody (Cat. No. NB300-221, Novus Biologicals).

Techniques: Binding Assay, Labeling, In Silico, Expressing, Western Blot, SDS Page, Membrane, Control

Journal: Scientific Reports

Article Title: UGT8/GalCer-dependent resistance of breast cancer cells to drug-induced apoptosis is potentially regulated by the LIM/homeobox protein LHX6

doi: 10.1038/s41598-026-42260-1

Figure Lengend Snippet: ( A ) Analysis of UGT8 mRNA expression levels (I), UGT8 protein levels (II), and GalCer levels (III) in wild-type MDA-MB-231 cells, MDA-MB-231 cells transfected with scrambled shRNA (MDA.C), and MDA-MB-231 cells transfected with shRNA directed against LHX6 mRNA (MDA.shLHX6). qPCR was used to analyze UGT8 mRNA. Its expression levels were normalized against GAPDH, and MDA.shLHX6 cells were used as a calibrator sample. All values are mean ± SD of at least 2 independent experiments, each assayed in triplicate. *** p < 0.001. UGT8 protein expression was analyzed by Western blotting. Rabbit polyclonal antibodies directed against UGT8 were used to detect UGT8. GalCer was analyzed by immunostaining of neutral GSLs separated by HP-TLC, with rabbit polyclonal anti-GalCer antibodies. For immunostaining, aliquots of total neutral GSLs corresponding to 1 × 10 7 cells were applied to an HP-TLC plate. ( B ) Sensitivity of MDA-MB-231 cells treated with shRNA directed against LHX6 mRNA to apoptosis induced by doxorubicin. Cells were grown in the presence of doxorubicin at concentration of 0.5 μM for 48 h. Cellular response measured by staining with Annexin V and SYTOX Green. Flow cytometry dot plots show percentage of early apoptotic cells (Annexin V + /SYTOX Green − , lower right) and late apoptotic cells (Annexin V + /SYTOX Green + , upper right). Dot blots display representative results obtained from two independent experiments. ( C ) Positive correlation between UGT8 and LHX6 protein expression levels in invasive ductal carcinoma (IDC) (immunohistochemistry, IHC). r = 0.46, p < 0.001 (Spearman’s correlation). ( D ) IHC LHX6 and UGT8 expression in IDC tissues with regard to pT, * p < 0.05; ** p < 0.01. ( E ) IHC LHX6 and UGT8 expression in IDC tissues with regard to pN. ( F ) IHC LHX6 and UGT8 expression in IDC tissues with regard to clinical stadium, * p < 0.05; ** p < 0.01.

Article Snippet: To detect specific proteins, the membrane slices were incubated with appropriate primary antibodies: anti-UGT8 (Cat. No. orb214717, Biorbyt), anti-GSX1 (Cat. No. BS-11612R, Thermo), anti-SOX4 (Cat. No. PA595290 , Thermo), anti-LHX6 (Cat. No. orb1535192, Biorbyt) and mouse monoclonal anti-GAPDH antibody (Cat. No. NB300-221, Novus Biologicals).

Techniques: Expressing, Transfection, shRNA, Western Blot, Immunostaining, Concentration Assay, Staining, Flow Cytometry, Immunohistochemistry, Immunohistochemistry-Resin

Journal: Cell & Bioscience

Article Title: The cytoskeleton regulates cytoophidium dynamics in Drosophila ovaries

doi: 10.1186/s13578-026-01530-1

Figure Lengend Snippet: Microtubules are essential for cytoophidium morphological changes, movement and assembly. A Time-lapse imaging revealed cytoophidia morphological changes, The time marked in the upper left corner represents the time since the capture began. The CTPS (gray) signal shown is obtained using mCherry-tagged CTPS. Scale bar = 5 μm. B Cytoophidia abundance and length are affected by microtubule depolymerization. Microtubules (green) are labeled with FITC-conjugated tubulin antibody. CTPS (red) signal is obtained using mCherry-tagged CTPS. scale bars, 20 μm. C Quantification of cytoophidium intercellular transportation through nurse cell-oocyte ring canals in control and microtubule inhibited groups. We analyzed 25 ovaries in control group and 25 ovaries in microtubule depolymerization group. D The speed of cytoophidia movement. We analyzed 35 filaments in control group and 35 filaments in microtubule depolymerization group. Data are represented as mean ± SEM. *** p < 0.001. E Length of cytoophidia in ovary nurse cell. We analyzed 32 filaments in control group and 88 filaments in microtubule depolymerization. Data are represented as mean ± SEM. *** p < 0.001. F Number of cytoophidia in ovary nurse cell. We analyzed 9 ovaries in control group and 413 filaments in total, and 9 ovaries in microtubule depolymerization and 1417 filaments in total, Data are represented as mean ± SEM. ** p < 0.001. G Western blotting analysis of CTPS proteins from ovary of CTPS-mCherry fly. Anti-mCherry antibody was used for the immunoblotting analysis. GAPDH was used as an internal control. H The CTPS protein levels in the samples from G were quantified. Data are represented as mean ± SEM. ns, no significance in difference. I Measurement of the relative CTPS mRNA expression was conducted via quantitative RT-PCR using ovary from the control and MT inhibitor-treated flies (6 ovaries/group, 3 biological replicates). Data are represented as mean ± SEM. ns, no significance in difference

Article Snippet: It was then incubated with the following primary antibodies: anti-mCherry tag monoclonal antibody (Abbkine, A02080) to label CTPS, and anti-GAPDH monoclonal antibody (Novus, NB300-221SS) as the internal control.

Techniques: Imaging, Labeling, Control, Western Blot, Expressing, Quantitative RT-PCR