Journal: Journal of Virology

Article Title: The African swine fever virus MGF360-16R protein functions as a mitochondrial-dependent apoptosis inducer by competing with BAX to bind to the HSP60 protein

doi: 10.1128/jvi.01401-24

Figure Lengend Snippet: MGF360-16R protein activates BAX by competing with BAX to bind to HSP60 protein. ( A ) WSL was transfected with either an empty vector (pcDNA3.1) or its recombinant plasmids expressing Myc-tagged MGF360-16R or truncation 1–145 aa for 36 h. The cell lysates were analyzed by Co-IP assays using anti-Myc magnetic beads and with the antibodies against HSP60, YWHAB, YWHAE, BAX, MGF360-16R, and GAPDH. A mouse isotype IgG1 was used as a control. ( B ) WSL was transfected as described in ( A ) for 24 h. The cells were analyzed by confocal immunofluorescence using antibodies against MGF360-16R and HSP60. ( C ) Co-IP analysis of the interaction between endogenous HSP60 and MGF360-16R in ASFV-infected primary PAMs at 48 hpi (MOI = 2). A rabbit isotype IgG was used as a control. ( D ) Confocal immunofluorescence analysis of the interaction between endogenous HSP60 and MGF360-16R in ASFV-infected WSL at 24 hpi (MOI = 2). The enlarged image of the area within the white-dashed box is provided on the right. ( E ) WSL was co-transfected with recombinant plasmids expressing Myc-tagged MGF360-16R and Flag-tagged HSP60 or their respective empty vectors (vector 1: pcDNA3.1-Myc; vector 2: pcDNA3.1-Flag) for 36 h. The cell lysates were analyzed by immunoblotting using antibodies against MGF360-16R, Flag, PARP1, CASP3, BAX, and β-actin. ( F ) WSL was co-transfected with recombinant plasmids expressing Myc-tagged MGF360-16R, Flag-tagged HSP60, and HA-tagged BAX or their respective empty vectors (vector 1: pcDNA3.1-Myc; vector 2: pcDNA3.1-Flag; vector 3: pCMV-HA) for 36 h. The cell lysates were analyzed by Co-IP assays using anti-Flag magnetic beads and with the antibodies against MGF360-16R, HA, Flag, and GAPDH. Representative results ( A, C, E, F ) from experiments performed in three independent biological replicates are shown.

Article Snippet: Mouse anti-HA (M180) and Flag (M185) mAbs were purchased from MBL Beijing Biotech Co., Ltd. Rabbit anti-GAPDH (10494–1-AP), PARP1 (13371–1-AP), YWHAE (11648–2-AP), HSP60 (15282–1-AP), GFP (50430–2-AP), Tom20 (11802–1-AP) and Myc (16286–1-AP) pAbs, and mouse anti-Myc (60003–2-Ig), YWHAB (66061–1-Ig), BAX (60267–1-Ig), β-actin (66009–1-Ig) mAbs were purchased from Proteintech Group, Inc. Rabbit anti-caspase-3 (14220), cleaved caspase-3 (9664), Cyt c (11940), COX IV (4850), and MitoTracker Red CMXRos 568 (9082) were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Transfection, Plasmid Preparation, Recombinant, Expressing, Co-Immunoprecipitation Assay, Magnetic Beads, Control, Immunofluorescence, Infection, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: YWHAB knockdown inhibits cell proliferation whilst promoting cell cycle arrest and apoptosis in colon cancer cells through PIK3R2

doi: 10.3892/etm.2023.11892

Figure Lengend Snippet: YWHAB can bind to PIK3R2 in colon cancer cells. (A) Monarch Initiative database predicted that YWHAB can bind to PIK3R2. (B) The mRNA expression of PIK3R2 was detected in colon cancer cell lines by RT-qPCR. (C) The binding of YWHAB and PIK3R2 was verified by co-immunoprecipitation assay. PIK3R2 (D) mRNA and (E) protein expression were detected by RT-qPCR and western blotting. *** P<0.001. YWHAB, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein β; PIK3R2, PI3K regulatory subunit 2; RT-qPCR, reverse transcription-quantitative PCR; shRNA, short hairpin RNA; NC, negative control.

Article Snippet: For immunoprecipitation, the extracts were incubated with 2 μg YWHAB (cat. no. A71754-050; 1:100; EpiGentek Group, Inc.) or PIK3R2 (cat. no. ab180967; 1:80; Abcam) antibodies overnight at 4 ̊C.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Activation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YWHAB knockdown inhibits cell proliferation whilst promoting cell cycle arrest and apoptosis in colon cancer cells through PIK3R2

doi: 10.3892/etm.2023.11892

Figure Lengend Snippet: YWHAB regulates cell proliferation and cell cycle arrest in colon cancer cells by interacting with PIK3R2. The expression of PIK3R2 (A) mRNA and (B) protein in transfected cells was detected by reverse transcription-quantitative PCR and western blotting. *** P<0.001. Un-transfected HCT116 cells were used as the Control. (C) Cell proliferation was detected by Cell Counting Kit-8 assay. (D) Cell cycle progression was detected by flow cytometry and (E) quantified. (F) The expression of cell cycle markers was detected by western blotting. *** P<0.001 vs. shRNA-NC; ## P<0.01 and ### P<0.001 vs. shRNA-YWHAB + Ov-NC. YWHAB, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein β; PIK3R2, PI3K regulatory subunit 2; shRNA, short hairpin RNA; NC, negative control; Ov, overexpressing vector.

Article Snippet: For immunoprecipitation, the extracts were incubated with 2 μg YWHAB (cat. no. A71754-050; 1:100; EpiGentek Group, Inc.) or PIK3R2 (cat. no. ab180967; 1:80; Abcam) antibodies overnight at 4 ̊C.

Techniques: Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control, Cell Counting, Flow Cytometry, shRNA, Activation Assay, Negative Control, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: YWHAB knockdown inhibits cell proliferation whilst promoting cell cycle arrest and apoptosis in colon cancer cells through PIK3R2

doi: 10.3892/etm.2023.11892

Figure Lengend Snippet: YWHAB regulates colon cancer cell apoptosis by binding to PIK3R2. (A) Cell apoptosis was detected using TUNEL staining. (B) The expression of apoptosis regulators was detected by western blotting. *** P<0.001 vs. Control; ## P<0.01 and ### P<0.001 vs. shRNA-YWHAB + Ov-NC. YWHAB, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein β; PIK3R2, PI3K regulatory subunit 2; shRNA, short hairpin RNA; NC, negative control; Ov, overexpressing vector.

Article Snippet: For immunoprecipitation, the extracts were incubated with 2 μg YWHAB (cat. no. A71754-050; 1:100; EpiGentek Group, Inc.) or PIK3R2 (cat. no. ab180967; 1:80; Abcam) antibodies overnight at 4 ̊C.

Techniques: Binding Assay, TUNEL Assay, Staining, Expressing, Western Blot, Control, shRNA, Activation Assay, Negative Control, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: YWHAB knockdown inhibits cell proliferation whilst promoting cell cycle arrest and apoptosis in colon cancer cells through PIK3R2

doi: 10.3892/etm.2023.11892

Figure Lengend Snippet: YWHAB regulates the PI3K/AKT signaling pathway by binding to PIK3R2. The expression of PI3K/AKT signaling marker proteins was detected by western blotting. *** P<0.001 vs. Control; # P<0.05 and ### P<0.001 vs. shRNA-YWHAB + Ov-NC. YWHAB, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein β; PIK3R2, PI3K regulatory subunit 2; shRNA, short hairpin RNA; NC, negative control; Ov, overexpressing vector; p-, phosphorylated; t-, total.

Article Snippet: For immunoprecipitation, the extracts were incubated with 2 μg YWHAB (cat. no. A71754-050; 1:100; EpiGentek Group, Inc.) or PIK3R2 (cat. no. ab180967; 1:80; Abcam) antibodies overnight at 4 ̊C.

Techniques: Binding Assay, Expressing, Marker, Western Blot, Control, shRNA, Activation Assay, Negative Control, Plasmid Preparation