Journal: RSC Advances

Article Title: Apoptosis induction and cell cycle arrest induced by Sinkiangenone B, a novel phenylpropanoid derivative from the resin of Ferula sinkiangensis K. M. Shen

doi: 10.1039/c7ra13716h

Figure Lengend Snippet: Fig. 8 The effects of Sinkiangenone B on the expression of apoptosis-related proteins determined by western blot. AGS cells were treated with Sinkiangenone B (0, 5, 10, 15 mm) for 24 h. Sinkiangenone B decreased the expression of Bcl-2 and cleaved PARP, and increased the expression of Bax and cleaved caspase-3. Relative expression levels of apoptosis-related proteins were showed. b-Tubulin was used to confirm equal protein loading. *p < 0.05 and **p < 0.01 were considered statistically significant.

Article Snippet: Antibodies against Bax, Bcl-2, cleaved PARP, cleaved caspase-3, cyclin D1, cyclin E, Cdk4, Cdk2, P16, P27 and P53 were purchased from Santa Cruz Biotechnology RSC Adv., 2018, 8, 4093–4103 | 4101 O pe n A cc es s A rt ic le .

Techniques: Expressing, Western Blot

Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 enhances oxaliplatin resistance in gastric cancer cells in vitro . Effects of oxaliplatin and fluorouracil supplementation on DEPP1 protein levels in (A) MKN45 and (B) HGC27 cells, respectively. Validation of DEPP1 overexpression in (C) MKN45 and (D) HGC27 cells. (E) Impact of DEPP1 overexpression on MKN45 cell proliferation, assessed using EdU flow cytometry. (F) Influence of DEPP1 overexpression on MKN45 proliferation following a 24-h treatment with 10 µM oxaliplatin. (G) Effects of ectopic DEPP1 expression on oxaliplatin-induced apoptosis (20 µM) in MKN45 cells, as measured by Annexin V/PI flow cytometry. (H) Analysis of full-length and cleaved PARP protein levels following forced DEPP1 expression in (H) MKN45 and (J) HGC27 cells. *P<0.05 and ***P<0.001 DEPP1, decidual protein induced by progesterone. ns, not significant.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: In Vitro, Biomarker Discovery, Over Expression, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Ubiquitin Ligase RNF146 Regulates Tankyrase and Axin to Promote Wnt Signaling

doi: 10.1371/journal.pone.0022595

Figure Lengend Snippet: (A) Western analysis of auto-ubiquitylation reactions with variable amounts of GST-RNF146 protein (see ) immunoblotted for flag-ubiquitin, polyubiquitin, or GST. GST protein serves as a negative control. (B) Western analysis as in (A) for GST-RNF146 ubiquitylation reactions incubated in the absence (−) or presence (+) of poly(ADP-ribose) [PAR] and immunoprecipitated with antibodies specific for K11-, K48-, or K63-linked polyubiquitin. Immunoblotting with RNF146 antibody serves as a loading control. Note that the K48 linkage-specific antibody more efficiently immunoprecipitates ubiquitylated RNF146, although the polyubiquitin chains are shorter in length and therefore less readily detected by anti-flag or -ubiquitin immunoblotting. (C) Coomassie-stained gel of anti-HA immunoprecipitates from cells transfected with the indicated expression constructs for RNF146 or control E3 ligase HECTD1. Numbered protein bands and lettered high-molecular-weight bands were excised for mass spectrometric analysis. (D) Table of proteins identified from the mass spectrometric analysis showing total numbers of peptides identified for each protein, combined for all numbered protein bands in (C). Shown are 23 proteins with the highest number of total peptides identified by interaction with RNF146ΔRING protein, and with fewer than three peptides in the spectrometric analysis of HECTD1 protein interactors. The second set of 5 proteins in the table show the greatest numbers of peptides identified for the analysis of interactors with wildtype RNF146 protein, but not HECTD1 protein. The code for the coloring is explained in the legend. (E) Western analysis of anti-V5 immunoprecipitation from HEK293 cells co-transfected as indicated for expression of V5-tagged wildtype or H53A mutant RNF146 (RNF), HA-tagged TNKS2, or control vector in the presence (+) and absence (−) of proteasome inhibitor ALLN. Co-immunoprecipitation of endogenous TNKS1 and overexpressed TNKS2 was assessed with anti-TNKS1/2 antibodies from the indicated two sources. RNF146 immunoblotting, anti-HA immunoprecipitation, and whole cell lysates are shown as controls. (F) Western analysis of immunoprecipitation of flag-tagged AXIN1 expressed in HEK293 cells co-transfected with the indicated expression constructs for HA-tagged wildtype or deletion mutant alleles of RNF146, TNKS1, or PARP1. Short exposure to film of the anti-HA immunoblot detects co-immunoprecipitated TNKS1 proteins, whereas longer exposure reveals RNF146 and PARP1 proteins. Input whole cell lysates probed for HA and flag detection are shown as controls for expression of the indicated proteins.

Article Snippet: An HCT-15 cell line stably integrated with TOPbrite reporter was selected for hygromycin resistance, and luciferase activity for these cells was normalized to AFC fluorescence with the CellTiter-Fluor cell viability assay (Promega). cDNAs for human RNF146 (NM_030963), TNKS1 (NM_003747), TNKS2 (NM_025235), and PARP1 (NM_001618) were purchased from OriGene and subcloned into pRK mammalian expression vectors.

Techniques: Western Blot, Negative Control, Incubation, Immunoprecipitation, Staining, Transfection, Expressing, Construct, Molecular Weight, Mutagenesis, Plasmid Preparation

Journal: PLoS ONE

Article Title: Ubiquitin Ligase RNF146 Regulates Tankyrase and Axin to Promote Wnt Signaling

doi: 10.1371/journal.pone.0022595

Figure Lengend Snippet: (A) Western analysis of anti-ubiquitin (FK2 antibody) immunoprecipitation from HEK293 cells transfected with expression constructs for wildtype or H53A mutant RNF146 (RNF) or control E3 ligase AMFR, in combination with either TNKS2 or control vector DNA, without (−) or with (+) proteasome inhibitor ALLN treatment. Immunoblots are shown for ubiquitylated tankyrase and RNF146 smears. Input lysate immunoblots show expression levels of tankyrase and RNF146. (B) Western analysis analogous to (A) except that HA-tagged PARP1 is overexpressed rather that TNKS2. RNF146 binding to PARP1 is confirmed by anti-HA immunoprecipitation. (C) Relative quantitation of the area under the mass spectra curve (AUC) for K48-, K63-, and K11-linked polyubiquitin -GG signature peptides in excised gel bands A, B, and C depicted in . Results are shown for immunoprecipitation of wildtype or H53A mutant RNF146 proteins expressed in HEK293 cells. (D) Western analysis of immunoprecipitation with K48 or K63 linkage-specific polyubiquitin antibodies for the indicated overexpression of RNF146, tankyrase, or Axin in HEK293 cells. Immunoblotting for tankyrase, Axin, or control β-catenin proteins detects high-molecular-weight polyubiquitylated protein species.

Article Snippet: An HCT-15 cell line stably integrated with TOPbrite reporter was selected for hygromycin resistance, and luciferase activity for these cells was normalized to AFC fluorescence with the CellTiter-Fluor cell viability assay (Promega). cDNAs for human RNF146 (NM_030963), TNKS1 (NM_003747), TNKS2 (NM_025235), and PARP1 (NM_001618) were purchased from OriGene and subcloned into pRK mammalian expression vectors.

Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing, Construct, Mutagenesis, Plasmid Preparation, Binding Assay, Quantitation Assay, Over Expression, Molecular Weight