primary antibodies against prohibitin-2 (phb2)  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: PHB2 promotes colorectal cancer cell proliferation and tumorigenesis through NDUFS1-mediated oxidative phosphorylation

doi: 10.1038/s41419-023-05575-9

Figure Lengend Snippet: A , B The expression of PHB2 in CRC and adjacent tissues was analyzed by the TCGA database. C Representative microphotographs of IHC and H&E staining of PHB2 (brown) on adjacent tissues ( n = 26), precancerous lesions ( n = 14), and CRC stage I–II ( n = 11) and stage III–IV ( n = 11) tissue sections. D Quantification analysis of PHB2 expression in precancerous lesions, CRC, and adjacent tissues. E The PHB2 expression levels in tissues of colorectal adenomatous polyps (APs) ( n = 2), CRC ( n = 4), and control patients ( n = 2) were detected by western blot. F Whole-cell lysates from a panel of CRC cells and normal intestinal epithelial cells were subjected to western blot. G Total RNA from a panel of CRC cells and normal intestinal epithelial cells were subjected to qRT-PCR. The relative abundance of PHB2 mRNA expression in NCM460 was arbitrarily designated as 1 ( n = 3). Mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The specific primary antibodies against PHB2 (1:2000), β-actin (1:5000), Flag tag (1:1000), NDUFS1 (1:2000), HA tag (1:4000), GST tag (1:5000), and NDUFV1 (1:1000) were purchased from Proteintech Group.

Techniques: Expressing, Staining, Control, Western Blot, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: PHB2 promotes colorectal cancer cell proliferation and tumorigenesis through NDUFS1-mediated oxidative phosphorylation

doi: 10.1038/s41419-023-05575-9

Figure Lengend Snippet: Lentivirus encoding negative control shRNA or PHB2 shRNA was transduced into HCT116 and HT-29 cells. A Western blot analysis of the whole cell lysates. B Cell viability was measured by MTT assays. C Representative images and quantitation of clonogenic assays. D Cell proliferation was detected by BrdU incorporation assays. E SW620 and NCM460 cells were transiently transfected with empty vector or Flag-PHB2 plasmids for 48 h. The whole cell lysates were subjected to western blot. F , G Cell viability and proliferation of SW620 cells transfected with Flag-PHB2 plasmids were measured by MTT assays ( F ) and BrdU incorporation assays ( G ). H Representative images and quantitation of anchorage-independent growth of NCM460 cells transfected with Flag-PHB2 plasmids. Mean ± SEM, * p < 0.05, ** p < 0.01.

Article Snippet: The specific primary antibodies against PHB2 (1:2000), β-actin (1:5000), Flag tag (1:1000), NDUFS1 (1:2000), HA tag (1:4000), GST tag (1:5000), and NDUFV1 (1:1000) were purchased from Proteintech Group.

Techniques: Negative Control, shRNA, Western Blot, Quantitation Assay, BrdU Incorporation Assay, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: PHB2 promotes colorectal cancer cell proliferation and tumorigenesis through NDUFS1-mediated oxidative phosphorylation

doi: 10.1038/s41419-023-05575-9

Figure Lengend Snippet: A Representative images of xenograft tumors. B Comparison of tumors weights in animals implanted with HT-29.sh-scramble and HT-29.sh-PHB2 cells ( n = 6). C The growth curves of xenograft tumors are presented. Tumor volume was calculated with a modified ellipsoidal formula ( n = 6). D The whole-cell lysates of crude tumor tissues from three randomly sampled tumors from each group were subject to western blot. E Representative images of IHC staining with anti-Ki67 in xenograft tumors. F Quantification analysis of Ki67 expression in xenograft tumors. Mean ± SEM, * p < 0.05, ** p < 0.01.

Article Snippet: The specific primary antibodies against PHB2 (1:2000), β-actin (1:5000), Flag tag (1:1000), NDUFS1 (1:2000), HA tag (1:4000), GST tag (1:5000), and NDUFV1 (1:1000) were purchased from Proteintech Group.

Techniques: Comparison, Modification, Western Blot, Immunohistochemistry, Expressing

Journal: Cell Death & Disease

Article Title: PHB2 promotes colorectal cancer cell proliferation and tumorigenesis through NDUFS1-mediated oxidative phosphorylation

doi: 10.1038/s41419-023-05575-9

Figure Lengend Snippet: A Mitochondrial OXPHOS activity in control, PHB2-deficient HCT116 and HT-29 cells were measured by Seahorse Bioscience XF96 extracellular flux analyzer. After establishing a baseline, oligomycin (1 μM), FCCP (1.5 μM), rotenone (0.5 μM), and antimycin A (0.5 μM) were sequentially added, as indicated by arrows. B , C Quantification of basal respiration and ATP production. D ATP content of HCT116 and HT-29 cells infected with shRNA against PHB2 was indicated by absorbance change measured at 340 nm. E , F Mitochondrial ROS levels were analyzed by confocal microscopy ( E ) and flow cytometry ( F ) after staining with MitoSOX™ (5 μM). Mean ± SEM, ** p < 0.01, *** p < 0.001.

Article Snippet: The specific primary antibodies against PHB2 (1:2000), β-actin (1:5000), Flag tag (1:1000), NDUFS1 (1:2000), HA tag (1:4000), GST tag (1:5000), and NDUFV1 (1:1000) were purchased from Proteintech Group.

Techniques: Activity Assay, Control, Infection, shRNA, Confocal Microscopy, Flow Cytometry, Staining

Journal: Cell Death & Disease

Article Title: PHB2 promotes colorectal cancer cell proliferation and tumorigenesis through NDUFS1-mediated oxidative phosphorylation

doi: 10.1038/s41419-023-05575-9

Figure Lengend Snippet: A Whole-cell lysates from HCT116 and HT-29 cells were subjected to immunoprecipitation with anti-PHB2 and anti-NDUFS1 antibodies. The Immunoglobulin G (IgG) was used as a negative control. B Whole-cell lysates from NCM460 cells co-transfected with Flag-PHB2 and HA-NDUFS1 plasmids were subjected to anti-Flag and anti-HA immunoprecipitation, respectively. C Top: Recombinant GST-PHB2 pulls down NDUFS1 from the cell lysates of HCT116 and HT-29 cells. Bottom: Western blot analysis of purified recombinant GST proteins from a pull-down assay. D Representative immunofluorescence images showing the co-localization of PHB2 (blue), NDUFS1 (green) and mitochondria (red) in HCT116 and HT-29 cells. E Western blot analysis of the whole cell lysates. F Analysis of complex I activity in HCT116 and HT-29 cells infected with shRNA against PHB2. Mean ± SEM, ** p < 0.01.

Article Snippet: The specific primary antibodies against PHB2 (1:2000), β-actin (1:5000), Flag tag (1:1000), NDUFS1 (1:2000), HA tag (1:4000), GST tag (1:5000), and NDUFV1 (1:1000) were purchased from Proteintech Group.

Techniques: Immunoprecipitation, Negative Control, Transfection, Recombinant, Western Blot, Purification, Pull Down Assay, Immunofluorescence, Activity Assay, Infection, shRNA

Journal: Cell Death & Disease

Article Title: PHB2 promotes colorectal cancer cell proliferation and tumorigenesis through NDUFS1-mediated oxidative phosphorylation

doi: 10.1038/s41419-023-05575-9

Figure Lengend Snippet: A Mitochondrial DNA (mtDNA) content was measured by qPCR. B Western blot analysis of the whole cell lysates from NCM460 and SW620 cells transfected with Flag-PHB2 plasmids. C NCM460 and SW620 cells were transfected with Flag-PHB2 plasmids for 48 h, and then the cells were treated with rotenone at concentrations of 0, 25, 50, 100 nM, or IACS-10759 at concentrations of 0, 30, 60, 120 nM for 24 h. Complex I activity was measured by a microplate reader displaying absorbance at 340 nm. Empty vector transfection served as control. D , E NCM460 and SW620 cells overexpressed PHB2 were treated with rotenone (50 nM) or IACS-10759 (60 nM) for 24 h. Cell viability and proliferation were measured by MTT assays ( D ) and BrdU incorporation assays ( E ). F HCT116 and HT-29 cells infected with shRNA against PHB2 were subjected to immunoprecipitation with anti-NDUFS1 or anti-NDUFV1. G , H HCT116 and HT-29 cells were transfected with siRNA against NDUFS1. Whole cell lysates were subjected to western blot ( G ) and immunoprecipitation with anti-PHB2 or anti-NDUFV1 ( H ). I Schematic illustration of the potential mechanism by which elevated PHB2 expression promotes cell proliferation and tumorigenesis of CRC. Mean ± SEM, * p < 0.05, ** p < 0.01.

Article Snippet: The specific primary antibodies against PHB2 (1:2000), β-actin (1:5000), Flag tag (1:1000), NDUFS1 (1:2000), HA tag (1:4000), GST tag (1:5000), and NDUFV1 (1:1000) were purchased from Proteintech Group.

Techniques: Western Blot, Transfection, Activity Assay, Plasmid Preparation, Control, BrdU Incorporation Assay, Infection, shRNA, Immunoprecipitation, Expressing