mouse monoclonal antibody against gapdh  (Proteintech)

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Desialylation results in the activation of the Hippo pathway. A, MDA-MB-231 cells were pretreated with different doses of sialidase for 3 h; then, the cell membrane fractions were immunoblotted with SNA (recognizing α2,6-sialylated proteins) and ConA (an α-mannose/α-glucose-binding lectin) lectins or blotted with anti-integrin β1 antibody. B, to further determine the change of sialylation on the cell surface after sialidase treatment, the indicated cells were incubated with biotin-conjugated MAA (recognizing 2,3-sialylated proteins, dotted line ), biotin-conjugated SNA ( bold line ), or without ( gray shadow ) lectin, followed by incubation with appropriate Alexa Flour 647 conjugate and subjected to flow cytometry. C, MDA-MB-231 cells were treated as described in ( A ), and then the cell lysates were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, and anti-GAPDH antibodies. The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1, respectively) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01, ∗∗∗, p < 0.001 is determined by two-tail unpaired t test). SNA, Sambucus nigra; MAA, Maackia amurensis agglutinin; ConA, Concanavalin A; LATS, large tumor suppressor kinase; YAP, yes-associated protein.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Activation Assay, Membrane, Binding Assay, Incubation, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: Knockout of ST6GAL1 leads to phosphorylation and functional inactivation of YAP in breast cancer cells. A–C, cell lysates from the control (Con)-, ST6GAL1-KO1 (KO1), and ST6GAL1-KO2 (KO2)- MDA-MB-231 cells ( A ) and cell lysates from the control (Con)-, ST6GAL1-KO2 (KO)- and KO-ST6GAL1-rescue (Res)- MDA-MB-231 ( B ) and BT549 ( C ) cells were immunoblotted by anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, anti-ST6GAL1, and anti-GAPDH antibodies. D, the relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1, respectively) are presented as the mean ± SD ( n = 3 biological replicates, ∗∗∗∗, p < 0.0001 is determined by one-way ANOVA with Tukey's post hoc test). E, the expression and localization of YAP in Con- and ST6GAL1-KO- MDA-MB-231 cells. Con and KO cells were cultured on coverslips, and cells were fixed, permeabilized, and then visualized with YAP. Nuclei were stained with TO-PRO-3. The scale bar represents 20 μm. F, the luciferase activity in Con-, ST6GAL1-KO-, and ST6GAL1-Res- MDA-MB-231 cells. Cells were cotransfected with 8 × GTIIC luciferase and TK-Renilla luciferase reporters; the reporter activities were measured using a Dual-Luciferase Reporter Assay System. G, the mRNA expression of ANKRD1 , CTGF , and CYR61 genes in Con-, ST6GAL1-KO-, and ST6GAL1-Res- MDA-MB-231 cells were detected by quantitative real-time PCR (qPCR). Error bars in ( F ), and ( G ) are mean ± SD ( n = 3 biological replicates, ∗∗, p < 0.01; ∗∗∗, p < 0.001, and ∗∗∗∗, p < 0.0001 are determined by one-way ANOVA with Tukey's post hoc test). LATS, large tumor suppressor kinase; YAP, yes-associated protein; ST6GAL1, β-galactoside α2,6-sialyltransferase 1.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Knock-Out, Phospho-proteomics, Functional Assay, Control, Expressing, Cell Culture, Staining, Luciferase, Activity Assay, Reporter Assay, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: α2,3-sialylation regulates the Hippo pathway through competing with α2,6-sialylation. A, the cell lysates from ST3GAL4-3xFLAG-overexpressing (ST3GAL4-OE) MDA-MB-231 cells were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, anti-FLAG, anti-ST6GAL1, and anti-GAPDH antibodies. B, the cell membrane fractions from Con and ST3GAL4-OE cells were blotted with ConA, RCA-I (recognizing terminal galactose), MAA, and SNA lectins. C, cell lysates from the MDA-MB-231 derivative cell lines as indicated (The ST6GAL1 KO + ST3GAL4 OE stable cell line was established by overexpressing ST3GAL4 in ST6GAL1 KO cells.) were immunoblotted with indicated antibodies as mentioned in ( A ). The relative ratios (phospho-YAP and phospho-LATS1 versus YAP and LATS1) in ( A ) and ( C ) are shown as the mean ± SD ( n = 3 biological replicates, n.s. not statistically significant, p > 0.05, ∗∗∗, p < 0.001, ∗∗∗∗, p < 0.0001 are determined by two-tail unpaired t test and one-way ANOVA with Tukey's post hoc test, respectively). D, the cell membrane fractions from the cells mentioned in ( C ) were blotted with ConA, RCA-I, MAA, and SNA lectins. LATS, large tumor suppressor kinase; SNA, Sambucus nigra ; YAP, yes-associated protein; RCA-I, Ricinus communis agglutinin I; ConA, Concanavalin A; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; MAA, Maackia amurensis agglutinin; Con, control.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Membrane, Stable Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Inhibitory effects of β-galactoside α2,6-sialyltransferase 1 on the Hippo pathway in breast cancer cells

doi: 10.1016/j.jbc.2025.110266

Figure Lengend Snippet: ST6GAL1–YAP axis regulates the metastatic ability of breast cancer cells. A, the cell lysates from Con- and ST6GAL1-OE- MDA-MB-231-Luc cells were immunoblotted with anti-p-YAP S127, anti-YAP, anti-p-LATS1 T1079, anti-LATS1, anti-ST6GAL1, and anti- GAPDH antibodies. The cell membrane fractions from the indicated cells were blotted with ConA, RCA-I, and SNA lectins. B and C, comparison of cell growth ( B ) and migration ( C ) among Con-, ST6GAL1-OE-cells treated with DMSO or VP. The cell proliferation ability ( B ) and the migration ability toward FN ( C ) were detected. The error bars are presented as the mean ± SD ( n = 3 biological replicates, n.s. not statistically significant, p > 0.05; ∗∗, p < 0.01, ∗∗∗, p < 0.001 are determined by two-way ANOVA with Tukey's post hoc test in panel B or one-way ANOVA with Tukey's post hoc test in panel C ). D and E, bioluminescent imaging ( D ) and quantification of photon flux ( E ) of NSG mice with intravenous injection of Con- and ST6GAL1-OE- MDA-MB-231-Luc cells, followed by DMSO or VP treatment. Day 0: the day of tumor cell injection. F and G, bioluminescent imaging (F, lower panel ), quantification of photon flux (F, upper panel ), and H&E staining ( G ) of the lungs from mice described in ( D ). The scale bar represents 250 μm. The error bars in ( E ) and ( F ) are presented as the mean ± SD ( n = 5 mice per group, n.s. not statistically significant, p > 0.05; ∗∗∗∗, p < 0.0001 are determined by two-way ANOVA with Tukey's post hoc test in panel E or one-way ANOVA with Tukey's post hoc test in panel F ). LATS, large tumor suppressor kinase; YAP, yes-associated protein; ST6GAL1, β-galactoside α2,6-sialyltransferase 1; OE, overexpressed; ConA, Concanavalin A; RCA-I, Ricinus communis agglutinin I; VP, verteporfin; FN, fibronectin; SNA, Sambucus nigra ; Con, control.

Article Snippet: The experiments were performed using the following antibodies: Rabbit antibodies against p-YAP(S127) (#13008S), p-LATS1(T1079) (#8654S), LATS1 (#3477S), p-Src(Y416) (#2101S), p-FAK(Y397) (#8556S), FAK (#3285S), EGFR (#4267S), p-EGFR(Y1068) (#3777S), and integrin β1 (#9699S) were from Cell Signaling Technology; mouse mAb against GAPDH (#sc-365062), and β-actin (#sc-47778) were from Santa Cruz Biotechnology; mouse mAb against integrin α5 (610633) was from BD Biosciences; rabbit pAbs against LPAR4 (22165-1-AP) and mouse mAb against YAP (66900-1-Ig) were obtained from Proteintech; rabbit pAb against ST3GAL4 (NBP1-69565) was obtained from Novus Biologicals; mouse mAbs against FLAG (clone M2, #F3165) and Src (clone GD11, #05-184) were from Sigma; goat pAb against ST6GAL1 (AF5924) was from R&D Systems; mouse mAb against integrin β1 (P5D2) was from Developmental Studies Hybridoma Bank.

Techniques: Membrane, Comparison, Migration, Imaging, Injection, Staining, Control

Journal: Current Issues in Molecular Biology

Article Title: Protective Effect of Hepcidin on Sepsis-Associated Acute Kidney Injury via Activating the Nrf2/GPX4 Signaling Pathway

doi: 10.3390/cimb47090772

Figure Lengend Snippet: Hepcidin inhibits ferroptosis in SAKI via activation of nuclear translocation of Nrf2. ( A ) Nuclear Nrf2 and cytoplasmic Nrf2 protein bands in renal tissue by WB; ( B ) Relative Nrf2 levels (Lamin B1) in renal nuclear fractions ( n = 3 mice per group); ( C ) Relative Nrf2 levels (GAPDH) in renal cytoplasmic fractions( n = 3 mice per group); ( D ) Nuclear-to-cytoplasmic Nrf2 ratio in renal tissue ( n = 3 per group); ( E ) GPX4 protein bands in renal tissue by Western blot; ( F ) Relative GPX4 protein levels by Western blot (GAPDH) in renal tissue lysates ( n = 3 mice per group); ( G ) Nrf2 protein expression by IHC; ( H ) Quantitative analysis of Nrf2 protein IOD values by IHC ( n = 3 mice per group). Compared with the sham group, ns, no significant difference; ** p < 0.01, *** p < 0.001, **** p < 0.0001; Compared with the CLP+Hamp group, # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001. Hamp, hepcidin.

Article Snippet: After incubation in blocking buffer for 30 min, the PVDF membranes were incubated overnight at 4 °C with a mouse antibody against GPX4 (1:2000, 67763-1-Ig, Proteintech, Wuhan, China), a rabbit antibody against ACSL4 (1:10,000, 22401-1-AP, Proteintech, Wuhan, China), a rabbit antibody against Nrf2 (1:1000, A0674, ABclonal, Wuhan, China), a rabbit antibody against Lamin B1 (1:10,000, 12987-1-AP, Proteintech, Wuhan, China), and a mouse antibody against GAPDH (1:50,000, 60004-1-Ig, Proteintech, Wuhan, China).

Techniques: Activation Assay, Translocation Assay, Western Blot, Expressing