Journal:

Article Title: Molecular Characterization of the phaEC Hm Genes, Required for Biosynthesis of Poly(3-Hydroxybutyrate) in the Extremely Halophilic Archaeon Haloarcula marismortui ▿

doi: 10.1128/AEM.00953-07

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: To confirm this, we knocked out the phaEC Hh genes from H. hispanica ATCC 33960 (see Materials and Methods) and thus generated a phaEC -deleted strain named H. hispanica PHB-1.

Techniques: Plasmid Preparation, Mutagenesis, Expressing, Knock-Out

Journal: International journal of molecular sciences

Article Title: PHB2 Alleviates Neurotoxicity of Prion Peptide PrP 106-126 via PINK1/Parkin-Dependent Mitophagy.

doi: 10.3390/ijms242115919

Figure Lengend Snippet: Figure 4. PHB2 involves PrP106–126-induced mitophagy in primary neurons. (A) Cortical neurons were treated with PrP106–126 for the indicated periods, and then, the protein levels of PHB2 were analyzed using Western blotting. (B) Cortical neurons were transfected with shRNA-PHB2 (sh#1, sh#2) plasmids or shRNA-NC control plasmid before the incubation with PrP106–126 for 24 h. PHB2, TOMM20, and LC3B-II were analyzed using Western blotting. (C) Cortical neurons were transfected with different concentrations of flag-PHB2 vector or flag-PCMV control vector for 24 h and then incubated with PrP106–126 for another 24 h. PHB2, TOMM20, and LC3B-II proteins were analyzed using Western blotting, respectively. (D) Representative images of PrP106–126-induced mitophago- some (red) formation in PHB2-knockdown neurons or PHB2-overexpression neurons. Cortical cells were firstly transfected with shRNA-PHB2 (sh#1) and flag-PHB2, respectively, and 24 h later, these neurons were transfected with COX8-EGFP-mCherry. After 24 h, the neurons were subjected to the PrP106–126 treatment for another 24 h. Finally, the neurons were fixed for confocal microscopy.

Article Snippet: Rabbit polyclonal anti-prohibitin 2 (PHB2) (12295-1-AP) antibody, rabbit polyclonal antibeta actin (β-actin) antibody (20536-1-AP), and mouse monoclonal anti-GAPDH antibody (60004-1-Ig) were obtained from Proteintech Group (Chicago, IL, USA).

Techniques: Western Blot, Transfection, shRNA, Control, Plasmid Preparation, Incubation, Knockdown, Over Expression, Confocal Microscopy

Journal: International journal of molecular sciences

Article Title: PHB2 Alleviates Neurotoxicity of Prion Peptide PrP 106-126 via PINK1/Parkin-Dependent Mitophagy.

doi: 10.3390/ijms242115919

Figure Lengend Snippet: Figure 5. PHB2 is required for PINK1/Parkin-mediated mitophagy in PrP106–126-treated neu- rons. (A) Western blotting of PINK1 and Parkin proteins in mitochondria of PHB2-overexpression and PHB2-knockdown neurons with the PrP106–126 treatments (HSP60 served as loading control). (B) The colocalization between mitochondria (labeled with HSP60, red) and Parkin (green) in PHB2- knockdown and PHB2-overexpression neurons under the PrP106–126 treatment was observed using immunofluorescence imaging (nuclei, blue).

Article Snippet: Rabbit polyclonal anti-prohibitin 2 (PHB2) (12295-1-AP) antibody, rabbit polyclonal antibeta actin (β-actin) antibody (20536-1-AP), and mouse monoclonal anti-GAPDH antibody (60004-1-Ig) were obtained from Proteintech Group (Chicago, IL, USA).

Techniques: Western Blot, Over Expression, Knockdown, Control, Labeling, Imaging

Journal: International journal of molecular sciences

Article Title: PHB2 Alleviates Neurotoxicity of Prion Peptide PrP 106-126 via PINK1/Parkin-Dependent Mitophagy.

doi: 10.3390/ijms242115919

Figure Lengend Snippet: Figure 6. PHB2 attenuates PrP106–126-induced neuronal death. Primary neurons (A) or PHB2- knockdown neurons (B) or PHB2-overexpression neurons (C) were treated with PrP106–126, the cell viability were measured by using the CCK-8 assay. Bars represent means ± SD (n = 3). ** p < 0.01 indicate significant differences between the control groups. (D) Fluorescence images of cell apoptosis were detected using TUNEL assays (red) in control or treated cortical neurons (nuclei, blue).

Article Snippet: Rabbit polyclonal anti-prohibitin 2 (PHB2) (12295-1-AP) antibody, rabbit polyclonal antibeta actin (β-actin) antibody (20536-1-AP), and mouse monoclonal anti-GAPDH antibody (60004-1-Ig) were obtained from Proteintech Group (Chicago, IL, USA).

Techniques: Knockdown, Over Expression, CCK-8 Assay, Control, Fluorescence, TUNEL Assay

Journal: International journal of molecular sciences

Article Title: PHB2 Alleviates Neurotoxicity of Prion Peptide PrP 106-126 via PINK1/Parkin-Dependent Mitophagy.

doi: 10.3390/ijms242115919

Figure Lengend Snippet: Figure 7. Schematic representation of PHB2-mediated mitophagy and neuronal death inhibition under PrP106–126 stimulation. PrP106–126 accumulates in mitochondria and leads to mitochondrial damage, which stabilizes PINK1 and recruits Parkin to mitochondria to mediate mitophagy. IMM component PHB2 acts as a mitophagy receptor and plays a vital role in PINK1/Parkin-dependent mitophagy. Endogenous and exogenous PHB2 functions as prosurvival proteins by mediating mi- tophagy and suppressing neuronal death. EX-PHB2, exogenous PHB2; EN-PHB2, endogenous PHB2.

Article Snippet: Rabbit polyclonal anti-prohibitin 2 (PHB2) (12295-1-AP) antibody, rabbit polyclonal antibeta actin (β-actin) antibody (20536-1-AP), and mouse monoclonal anti-GAPDH antibody (60004-1-Ig) were obtained from Proteintech Group (Chicago, IL, USA).

Techniques: Inhibition

Journal: Journal of Biological Chemistry

Article Title: Regulation of Syk by Phosphorylation on Serine in the Linker Insert

doi: 10.1074/jbc.m110.164509

Figure Lengend Snippet: FIGURE 7. Interaction of Syk with 14-3-3. A, DT40 cells stably expressing Syk-EGFP were treated with DMSO (D), 5 g/ml anti-IgM (I), or 100 ng/ml PMA (P) for 30 min. Cell lysates were adsorbed to GST or GST-14-3-3 bound to glutathione-Sepharose. Bound Syk-EGFP was detected by Western blot- ting. B, DT40 cells lacking Syk (Syk-def) or stably expressing Syk-EGFP (Syk) or Syk-EGFP(S291A) (S291A) were treated with DMSO () or PMA () for 30 min. Lysates were adsorbed to immobilized GST (GST) or GST-14-3-3 (14). Syk-EGFP in the lysates of Syk-deficient cells (lane 1) or of cells expressing Syk-EGFP (lane 2) or Syk-EGFP(S291A) (lane 3) or bound to the resin was de- tected by Western blotting. The migration positions of Syk-EGFP proteins is indicated by the arrow. C, lysates from unstimulated cells described in panel B were adsorbed to resin containing GST-14-3-3 or GST-14-3-3. Bound Syk-EGFP was detected by Western blotting (arrow). FIGURE 8. Interaction of Syk with PHB1. A, shown is the sequence of PHB1-derived peptides identified by mass spectrometric analysis of pro- teins binding to immobilized Ser(P)-291-derived peptide. B, lysates from DG75 cells were incubated with biotinylated peptides corresponding to the sequence surrounding Ser-291 with Ser-291-phosphorylated (pS291) or not (S291) and adsorbed onto streptavidin-agarose beads. Bound proteins were analyzed by Western blotting (IB) with an antibody against PHB1. The mi- gration position of PHB1 is indicated by the arrowhead. C, lysates from Esch- erichia coli expressing GST-PHB1 were incubated with the Ser-291 or Ser(P)- 291-containing peptides and adsorbed onto streptavidin-agarose. Bound GST-PHB1 (arrowhead) was detected by Western blotting. D, PHB1 was im- munoprecipitated from lysates of DG75 cells pretreated with DMSO (D), 5 g/ml anti-human IgM (I), or 100 ng/ml PMA (P). Syk and PHB1 were de- tected in the lysates and immune complexes by Western blotting. PHB1 migrates just above the immunoglobulin light chain. A mock immunopre- cipitation performed in the absence of anti-PHB1 is also shown (Ctrl). E, DG75 cells were pretreated with DMSO or piceatannol (20 g/ml) for 30 min and then treated with DMSO (D) or 100 ng/ml PMA (P) for 30 min. PHB1 was immunoprecipitated from cell lysates. The presence of Syk in the im- mune complexes was detected by Western blotting. A mock immunopre- cipitation performed in the absence of anti-PHB1 is also shown (Ctrl).

Article Snippet: A cDNA coding for human PHB1 obtained from Origene was amplified by PCR and subcloned into the pGEX 4-T-2 plasmid for the bacterial expression of GST-PHB1.

Techniques: Stable Transfection, Expressing, Western Blot, Migration, Sequencing, Derivative Assay, Binding Assay, Incubation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Regulation of Syk by Phosphorylation on Serine in the Linker Insert

doi: 10.1074/jbc.m110.164509

Figure Lengend Snippet: FIGURE 9. Interaction of Syk and Syk(S291D) with PHB1. A, PHB1 was immunoprecipitated from lysates of U937 cells pretreated with 100 ng/ml PMA for 30 min. The presence of Syk (upper panels) or PHB1 (lower panels) in the immune complexes was detected by Western blotting. A mock immu- noprecipitation performed in the absence of anti-PHB1 is also shown (Ctrl). Migration positions of immunoglobulin heavy (HC) and light (LC) chains are indicated. B, PHB1 was immunoprecipitated from lysates of U937 cells (U937) or resting mouse spleen B cells (B cells) pretreated with DMSO (D) or 100 ng/ml PMA (P) for 30 min. The presence of Syk (upper and lower panels) or PHB1 (middle panels) in the lysates and immune complexes was detected by Western blotting. Migration positions of immunoglobulin heavy (HC) and light (LC) chains are indicated. C, DG75 B cells were transiently trans- fected with plasmids for the expression of GFP (Ctrl), Syk-EGFP(S291D) (S291D), Syk-EGFP(S291A) (S291A), or Syk-EGFP (WT). The level of expression in cell lysates was compared by Western blotting (IB) with an anti-Syk anti- body (lower panel). The presence of Syk fusion proteins in anti-PHB1 im- mune complexes was detected by Western blotting (WB) using an anti-GFP antibody (upper panel). The arrows mark the migration positions of Syk- EGFP fusion proteins.

Article Snippet: A cDNA coding for human PHB1 obtained from Origene was amplified by PCR and subcloned into the pGEX 4-T-2 plasmid for the bacterial expression of GST-PHB1.

Techniques: Immunoprecipitation, Western Blot, Migration, Expressing

Journal: iScience

Article Title: A LY6E-PHB1-TRIM21 assembly degrades CD14 protein to mitigate LPS-induced inflammatory response

doi: 10.1016/j.isci.2023.106808

Figure Lengend Snippet:

Article Snippet: Prohibitin Rabbit pAb , ABclonal , Cat# A0056; RRID: AB_2756921.

Techniques: Recombinant, Software, Membrane, Protein Extraction

Journal: Cancer Science

Article Title: Protein Profile of Multiple Myeloma‐Derived Extracellular Vesicles for the Discovery of Novel Myeloma‐Related Biomarkers

doi: 10.1111/cas.70473

Figure Lengend Snippet: Bioinformatic pipeline for EV‐related biomarker identification. To identify candidate MM EV–associated biomarkers, we applied a stepwise analytical workflow starting from functional enrichment and disease association analysis using the DisGeNET database to prioritize candidates with potential pathological relevance. Selected molecules were subsequently filtered based on their recurrence across disease‐associated datasets, leading to the identification of 21 candidate proteins. MM‐specific validation of these candidates was performed by evaluating them through MM‐specific transcriptomic datasets, including those curated by Neri and Gutierrez, followed by analysis of the MMRF CoMMpass cohort. Analysis through the database curated by Neri and Gutierrez ( n = 268 MM patients, 9 healthy donors and 18 HMCL) identified six molecules significantly deregulated in newly diagnosed MM compared with healthy donors, namely MIF, PHB, ANXA2, EZR, FN1 and KRAS. To assess their clinical relevance and prognostic impact, we analyzed the MMRF CoMMpass dataset ( n = 489 MM patients). Kaplan–Meier and Cox regression analyses revealed that high expression levels of MIF and PHB were significantly associated with shorter overall survival (OS) and progression‐free survival (PFS). Finally, selected candidate biomarkers were validated ex vivo in patient plasma samples by ELISA.

Article Snippet: Membranes were blocked in 5% nonfat milk in TBS‐T and probed overnight (4°C) with primary antibodies against ITGβ1 (1:4000, GTX128839; GenTex, USA), Flotillin2 (1:500, GTX114411; GenTex), DNMT1 (1:1000, sc‐20701; Santa‐Cruz Biotechnology, USA), HDAC2 (1:1000, sc‐7899; Santa‐Cruz Biotechnology), CDK6 (1:1000, D4S8S; Cell Signaling Technology, USA), Annexin11 (1:3000, ab300578; Abcam, USA), Histone 3 (1:5000, ab1791; Abcam), HSP90α (1:10000, GenTex), PHB (1:1000, A19530, Abclonal, Germany) and MIF (1:500, A22623; Abclonal).

Techniques: Biomarker Discovery, Functional Assay, Expressing, Ex Vivo, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: Protein Profile of Multiple Myeloma‐Derived Extracellular Vesicles for the Discovery of Novel Myeloma‐Related Biomarkers

doi: 10.1111/cas.70473

Figure Lengend Snippet: Evaluation of clinical significance of the gene's variability using MMRF CoMMpass Study. (A) Kaplan–Meier curves demonstrate the difference in overall survival (OS) and progress free survival (PFS) among 753 patients with MM stratified in high‐ and low‐expression groups based on the median expression level across the entire dataset for MIF (left) and PHB (right). Log‐rank test p ‐value measuring the global difference between survival curves and number of samples at risk in each group across time are reported. (B) Cox regression univariate analysis of OS (left) or PFS (right) data on MIF and PHB expression groups, age equal to or greater than 65 years, ISS subgroups and main molecular alterations in 489 BM MM cases for which all data were available. Number ( N ) of positive cases is indicated for each variable. Hazard Ratio, 95% confidence interval and Log‐rank p ‐value are reported for each variable. All significant variables after BH correction are depicted in red bold.

Article Snippet: Membranes were blocked in 5% nonfat milk in TBS‐T and probed overnight (4°C) with primary antibodies against ITGβ1 (1:4000, GTX128839; GenTex, USA), Flotillin2 (1:500, GTX114411; GenTex), DNMT1 (1:1000, sc‐20701; Santa‐Cruz Biotechnology, USA), HDAC2 (1:1000, sc‐7899; Santa‐Cruz Biotechnology), CDK6 (1:1000, D4S8S; Cell Signaling Technology, USA), Annexin11 (1:3000, ab300578; Abcam, USA), Histone 3 (1:5000, ab1791; Abcam), HSP90α (1:10000, GenTex), PHB (1:1000, A19530, Abclonal, Germany) and MIF (1:500, A22623; Abclonal).

Techniques: Expressing

Journal: Cancer Science

Article Title: Protein Profile of Multiple Myeloma‐Derived Extracellular Vesicles for the Discovery of Novel Myeloma‐Related Biomarkers

doi: 10.1111/cas.70473

Figure Lengend Snippet: Validation of candidate EV‐related biomarkers. (A) Western blot analysis for MIF and PHB on cell and vesicular lysates from a panel of 5 MM cell lines. The level of Annexin11 was used for normalization. Twenty μg of protein lysates were loaded per lane. (B, C) ELISA for MIF and PHB on plasma derived from HD (8/10), MGUS ( N = 10/12), SMM ( N = 16/19), and MM ( N = 17/19) newly diagnosed patients. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. * p < 0.05, *** p < 0.001. (D) ELISA of plasma depleted EV by ultracentrifugation for MIF on three independent plasma samples from patients with MGUS, SMM, MM, and HD. The results represent the percentage of MIF present in full plasma and after EV depletion by ultracentrifugation ± SEM. MIF in full plasma is settled as 100%.

Article Snippet: Membranes were blocked in 5% nonfat milk in TBS‐T and probed overnight (4°C) with primary antibodies against ITGβ1 (1:4000, GTX128839; GenTex, USA), Flotillin2 (1:500, GTX114411; GenTex), DNMT1 (1:1000, sc‐20701; Santa‐Cruz Biotechnology, USA), HDAC2 (1:1000, sc‐7899; Santa‐Cruz Biotechnology), CDK6 (1:1000, D4S8S; Cell Signaling Technology, USA), Annexin11 (1:3000, ab300578; Abcam, USA), Histone 3 (1:5000, ab1791; Abcam), HSP90α (1:10000, GenTex), PHB (1:1000, A19530, Abclonal, Germany) and MIF (1:500, A22623; Abclonal).

Techniques: Biomarker Discovery, Western Blot, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Derivative Assay