Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 2. Structure of the DB3 combining site. (A) Crystal structure of the complex between DB3 and progesterone (Arevalo et al. 1993a) showing some of the contact residues. Light and the heavy chains are in blue and purple, respectively; steroid oxygen atoms in red; steroid A-ring asterisked. (B) Model of site with TrpH100 replaced by arginine. (C) Superposition of A and B.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques:

Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 3. Western blot of wild type and mutants of DB3 VH/K. Samples were run on 10% SDS-PAGE and electrotransferred to an Immobilon-P membrane (PVDF); the blot was probed with HRP-coupled sheep anti- mouse k.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques: Western Blot, SDS Page, Membrane

Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 4. Antigen-binding and idiotypic properties of periplasmic extracts of E. coli expressing VH/K mutants. (A) Binding activity by ELISA on progesterone-BSA coated wells; (B) Competitive inhibition of binding by free progesterone (100 ng/mL) in ELISA; (C) Competitive inhibition by rabbit polyclonal anti-DB3-idiotype (10 mg/mL). Results are means of duplicates. (d) Deletion; (Co.) negative control.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques: Binding Assay, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Negative Control

Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 5. Steroid specificity of DB3 wild-type VH/K and TrpH100Arg mutant. Binding to progesterone-BSA of fixed amounts of wild-type DB3 VH/K (A) and the TrpH100Arg mutant (B) was carried out in the presence of free progesterone, aetiocholanolone, 5a-dihydroprogesterone, 5b-dihydroprogesterone, or testosterone as inhibitors. Results are means of duplicates.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques: Mutagenesis, Binding Assay

Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 6. Binding specificity of wild-type DB3 VH/K and the TrpH100Arg mutant for progesterone derivatives. Binding to progester- one-BSA of wild-type DB3 VH/K (A) and the TrpH100Arg mutant (B) was carried out in the presence of free progesterone-3-CMO (C3-CMO), progesterone-6-HMS (C6-HMS), progesterone-11a-HMS (C11a-HMS), or progesterone-21-HMS (C21-HMS). Results are means of duplicates.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques: Binding Assay, Mutagenesis

Journal: Frontiers in pharmacology

Article Title: Fruits of Hippophaë rhamnoides in human leukocytes and Caco-2 cell monolayer models-A question about their preventive role in lipopolysaccharide leakage and cytokine secretion in endotoxemia.

doi: 10.3389/fphar.2022.981874

Figure Lengend Snippet: FIGURE 5 Detection of GLUT2 translocation in Caco-2 cells in a fluorescence microscope. HR—aqueous extract from the fruit of Hipopphaë rhamnoides.

Article Snippet: Glut2 polyclonal antibody (600-401-GN3, Rockland, PA, United States), FITCconjugated goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (F-2765, Invitrogen, United Kingdom), and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; D1306) were purchased from Thermo Fisher Scientific (Rockford, IL, United States).

Techniques: Translocation Assay, Microscopy

Journal: Cancers

Article Title: SHARPIN Enhances Ferroptosis in Synovial Sarcoma Cells via NF-κB- and PRMT5-Mediated PGC1α Reduction.

doi: 10.3390/cancers15133484

Figure Lengend Snippet: Figure 4. SHARPIN enhances the sensitivity of synovial sarcoma cell lines to ferroptosis via the PGC1α/SLC7A11 axis. (A–D) Viability assays of Aska (A,B) and Yamato (C,D) cells expressing scrambled or SHARPIN-specific shRNAs and treated with the indicated concentration of RSL3 (A,C) or erastin (B,D) for 24 h. (E) Immunoblot analyses of the effects of knockdown of SHARPIN on the expression levels of PGC1α, SLC7A11, SHARPIN, complex I, III, V, VDAC1/3, Parkin, BNIP3L/NIX, and LC3B in Yamato and Aska cells. (F) A qPCR analysis of the effect of transient SMART- pool siRNA-mediated knockdown of SHARPIN on SLC7A11 mRNA expression in Yamato cells. (G) Immunoblot analyses of the effects of knockdown of SHARPIN on the expression levels of NRF2 in Yamato cells. (H) Complex I activity in Yamato cells expressing scrambled or SHARPIN-specific shRNAs. Cells were seeded in identical numbers and incubated overnight. Signal intensity was then measured at the indicated time points. (I) ROS assay of Yamato cells expressing scrambled or SHARPIN-specific shRNAs. The cells were treated with or without 0.01 µM RSL3 for 24 h prior to the measurement of ROS activity. (J) GSH/GSSG ratio assay of Yamato cells expressing scrambled or SHARPIN-specific shRNAs. (K) Analysis of the relationship between SHARPIN mRNA expression levels and the GPX4 dependency of a bone and soft tissue sarcoma cohort using Chronos, a dynamic model of CRISPR data (CCLE database). The population below the first quantile (n = 18) was regarded as the low group, the population between the first and third quantile (n = 33) was regarded as the middle group, and the population above the third quantile (n = 18) was regarded as the high group. (A–D,F) Quantitative data are presented as the mean ± SD (n = 3). (K) A box-and-whisker plot is shown. (A–D,I,J) Statistical significance was calculated using one- or two-way ANOVA. * p < 0.05; ** p < 0.005; *** p < 0.0005; NS, not significant. (F) Statistical significance was calculated using a

Article Snippet: Antibodies targeting the following proteins were used: TFRC (ab214039, Abcam, Cambridge, UK), FPN (NBP1-21502, Novus Biologicals, Englewood, CO, USA), FTH1 (ab65080, Abcam), SLC7A11 (600-401-GU3, Rockland Immunochemicals, Pottstown, PA, USA), GPX4 (ab125066, Abcam), GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), PGC1α (NBP1-04676, Novus Biologicals), SHARPIN (ABF128, Millipore, Burlington, MA, USA), β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), VDAC1/3 (ab14734, Abcam), PRMT5 (sc-376937, Santa Cruz Biotechnology), SDMA (SYM10; 07- 412, Millipore), SOX10 (sc-365692, Santa Cruz Biotechnology), MITF (ab12039, Abcam), LC3B (NB100-2220, Novus Biologicals), NRF2 (#12721, Cell Signaling Technology), Parkin (#4211, Cell Signaling Technology), and BNIP3L/NIX (#12396, Cell Signaling Technology).

Techniques: Expressing, Concentration Assay, Western Blot, Knockdown, Activity Assay, Incubation, ROS Assay, CRISPR, Whisker Assay

Journal: Cancers

Article Title: SHARPIN Enhances Ferroptosis in Synovial Sarcoma Cells via NF-κB- and PRMT5-Mediated PGC1α Reduction.

doi: 10.3390/cancers15133484

Figure Lengend Snippet: Figure 5. Aberrant PGC1α expression overwhelms the regulatory effect of SHARPIN inhibition on ferroptosis in CCS. (A) A qPCR analysis of PGC1α mRNA expression in several sarcoma or non-sarcoma cell lines including CCS cell lines (SU and KAS). (B) Immunoblot analyses of SHARPIN, PRMT5, SDMA, SOX10, MITF, PGC1α and SLC7A11 in four permanent CCS cell lines, one primary CCS cell line, and HDF. (C) A qPCR analysis of SHARPIN mRNA expression in CCS clinical samples (n = 11) and normal tissues (n = 4). (D) The effect of knockdown of SHARPIN on PGC1α protein expression in SU and KAS cell lines. (E) Viability assays of SU and KAS cells expressing scrambled or SHARPIN-specific shRNAs. The cells were treated with or without the indicated concentration of RSL3 for 24 h. Cell viability was measured using the ratio of live cells in treated/control. (C) A box-and-whisker plot is shown. Statistical significance was calculated using a Mann–Whitney U test. * p < 0.05. (E) Statistical significance was calculated via a one-way ANOVA or Student’s t-test. Quantitative data are presented as the mean ± SD (n = 3). NS, not significant. The uncropped blots are shown in File S1.

Article Snippet: Antibodies targeting the following proteins were used: TFRC (ab214039, Abcam, Cambridge, UK), FPN (NBP1-21502, Novus Biologicals, Englewood, CO, USA), FTH1 (ab65080, Abcam), SLC7A11 (600-401-GU3, Rockland Immunochemicals, Pottstown, PA, USA), GPX4 (ab125066, Abcam), GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), PGC1α (NBP1-04676, Novus Biologicals), SHARPIN (ABF128, Millipore, Burlington, MA, USA), β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), VDAC1/3 (ab14734, Abcam), PRMT5 (sc-376937, Santa Cruz Biotechnology), SDMA (SYM10; 07- 412, Millipore), SOX10 (sc-365692, Santa Cruz Biotechnology), MITF (ab12039, Abcam), LC3B (NB100-2220, Novus Biologicals), NRF2 (#12721, Cell Signaling Technology), Parkin (#4211, Cell Signaling Technology), and BNIP3L/NIX (#12396, Cell Signaling Technology).

Techniques: Expressing, Inhibition, Western Blot, Knockdown, Concentration Assay, Control, Whisker Assay, MANN-WHITNEY

Journal: Cancers

Article Title: SHARPIN Enhances Ferroptosis in Synovial Sarcoma Cells via NF-κB- and PRMT5-Mediated PGC1α Reduction.

doi: 10.3390/cancers15133484

Figure Lengend Snippet: Figure 6. PRMT5 and NF-κB are essential regulators of ferroptosis downstream of SHARPIN. (A) Immunoblot and qPCR analyses of the effect of knockdown of SHARPIN on the expression levels of SDMA protein and IL-6 mRNA in Yamato cells. (B) The effect of a PRMT5 inhibitor, EPZ01566, on PGC1α, SLC7A11, and SDMA protein levels in Yamato cells. (C) The effect of a NF-κB inhibitor, SC- 514, on PGC1α protein, SLC7A11 protein, and IL-6 mRNA levels in Yamato cells. (D) Kaplan–Meier curve showing the relationship between DFS and SHARPIN and/or TFRC gene amplification (AMP), based on a TCGA dataset of all types of cancer. Subjects were divided into SHARPIN only AMP, TFRC only AMP, SHARPIN and TFRC AMP, and no AMP groups. (E) Kaplan–Meier curve showing the relationship between OS and SHARPIN and/or TFRC gene amplification (AMP), based on a TCGA dataset of soft tissue sarcoma samples. Subjects were divided into SHARPIN only AMP or HIGH (z-score ≥2), TFRC only AMP or HIGH, SHARPIN and TFRC AMP or HIGH, and no AMP or HIGH groups. (A,C) Statistical significance was calculated via a one-way ANOVA or Student’s t-test. Quantitative data are presented as the mean ± SD (n = 3). * p < 0.05. (D,E) Statistical significance was calculated using a log-rank test. The p-value is shown in each figure. The uncropped blots are shown in File S1.

Article Snippet: Antibodies targeting the following proteins were used: TFRC (ab214039, Abcam, Cambridge, UK), FPN (NBP1-21502, Novus Biologicals, Englewood, CO, USA), FTH1 (ab65080, Abcam), SLC7A11 (600-401-GU3, Rockland Immunochemicals, Pottstown, PA, USA), GPX4 (ab125066, Abcam), GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), PGC1α (NBP1-04676, Novus Biologicals), SHARPIN (ABF128, Millipore, Burlington, MA, USA), β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), VDAC1/3 (ab14734, Abcam), PRMT5 (sc-376937, Santa Cruz Biotechnology), SDMA (SYM10; 07- 412, Millipore), SOX10 (sc-365692, Santa Cruz Biotechnology), MITF (ab12039, Abcam), LC3B (NB100-2220, Novus Biologicals), NRF2 (#12721, Cell Signaling Technology), Parkin (#4211, Cell Signaling Technology), and BNIP3L/NIX (#12396, Cell Signaling Technology).

Techniques: Western Blot, Knockdown, Expressing